RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy.

PubMed

Denkert, C; Loibl, S; Kronenwett, R; Budczies, J; von Törne, C; Nekljudova, V; Darb-Esfahani, S; Solbach, C; Sinn, B V; Petry, C; Müller, B M; Hilfrich, J; Altmann, G; Staebler, A; Roth, C; Ataseven, B; Kirchner, T; Dietel, M; Untch, M; von Minckwitz, G

2013-03-01

Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

Avian influenza virus detection and quantitation by real-time RT-PCR

USDA-ARS?s Scientific Manuscript database

Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

[Experimental studies of using real-time fluorescence quantitative PCR and RT-PCR to detect E6 and E7 genes of human papillomavirus type 16 in cervical carcinoma cell lines].

PubMed

Chen, Yue-yue; Peng, Zhi-lan; Liu, Shan-ling; He, Bing; Hu, Min

2007-06-01

To establish a method of using real-time fluorescence quantitative PCR and RT-PCR to detect the E6 and E7 genes of human papillomavirus type 16 (HPV-16). Plasmids containing HPV-16 E6 or E7 were used to generate absolute standard curves. Three cervical carcinoma cell lines CaSki, SiHa and HeLa were tested by real-time fluorescence quantitative PCR and RT-PCR analyses for the expressions of HPV-16 E6 and E7. The correlation coefficients of standard curves were larger than 0. 99, and the PCR efficiency was more than 90%. The relative levels of HPV-16 E6 and E7 DNA and RNA were CaSki>SiHa>HeLa cell. HPV-16 E6 and E7 quantum by real-time fluorescence quantitative PCR and RT-PCR analyses may serve as a reliable and sensitive tool. This study provides the possibility of further researches on the relationship between HPV-16 E6 or E7 copy number and cervical carcinoma.

A novel duplex real time quantitative reverse transcription polymerase chain reaction for rubella virus with armored RNA as a noncompetitive internal positive control.

PubMed

Zhao, Lihong; Li, Ruiying; Liu, Aihua; Zhao, Shuping

2015-07-01

The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system. Copyright © 2015 Elsevier B.V. All rights reserved.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong

2017-01-01

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

PubMed

Hommais, Florence; Zghidi-Abouzid, Ouafa; Oger-Desfeux, Christine; Pineau-Chapelle, Emilie; Van Gijsegem, Frederique; Nasser, William; Reverchon, Sylvie

2011-01-01

Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.

Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...

Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

USDA-ARS?s Scientific Manuscript database

A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

PubMed Central

Gunderson, Felizza F.; Cianciotto, Nicholas P.

2013-01-01

ABSTRACT Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of cas transcripts, especially those of cas1 and cas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells. PMID:23481601

Gene Profiling Technique to Accelerate Stem Cell Therapies for Eye Diseases

MedlinePlus

... like RPE. They also use a technique called quantitative RT-PCR to measure the expression of genes ... higher in iPS cells than mature RPE. But quantitative RT-PCR only permits the simultaneous measurement of ...

Use of a custom RT-PCR array to analyze toxicity pathways at different life stages in Brown Norway Rat Brain following acute Toluene exposure.

EPA Science Inventory

To investigate the contribution of different life stages on response to toxicants, we utilized a custom designed RT-PCR array to examine the effects of acute exposure by oral gavage of the volatile organic solvent toluene (0.00, 0.65 or 1.0 glkg) in the brains of ma1e Brown Norwa...

RT-qPCR Demonstrates Light-Dependent AtRBCS1A and AtRBCS3B mRNA Expressions in "Arabidopsis thaliana" Leaves

ERIC Educational Resources Information Center

Chang, Ming-Mei; Li, Anna; Feissner, Robert; Ahmad, Talal

2016-01-01

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it is necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to…

DNA methylation pattern of apoptosis-related genes in ameloblastoma.

PubMed

Costa, Sfs; Pereira, N B; Pereira, Kma; Campos, K; de Castro, W H; Diniz, M G; Gomes, C C; Gomez, R S

2017-09-01

DNA methylation is an important mechanism of gene control expression, and it has been poorly addressed in odontogenic tumours. On this basis, we aimed to assess the methylation pattern of 22 apoptosis-related genes in solid ameloblastomas. Ameloblastoma fresh samples (n = 10) and dental follicles (n = 8) were included in the study. The percentage fraction of methylated and unmethylated DNA promoter of 22 apoptosis-related genes was determined using enzymatic restriction digestion and quantitative real-time PCR (qPCR) array. The relative expressions of the genes that showed the most discrepant methylation profile between tumours and controls were analysed by reverse-transcription quantitative PCR (RT-qPCR). Lower methylation percentages of TNFRSF25 (47.2%) and BCL2L11 (33.2%) were observed in ameloblastomas compared with dental follicles (79.3% and 59.5%, respectively). The RT-qPCR analysis showed increased expression of BCL2L11 in ameloblastomas compared with dental follicles, in agreement with the methylation analysis results, while there was no difference between the expression levels of TNFRSF25 between both groups. On the basis of our results, the transcription of the apoptosis-related gene BCL2L11 is possibly regulated by promoter DNA methylation in ameloblastoma. The biological significance of this finding in ameloblastoma pathobiology remains to be clarified. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator

USDA-ARS?s Scientific Manuscript database

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, expres...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Science Inventory

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

Competitive RT-PCR Strategy for Quantitative Evaluation of the Expression of Tilapia (Oreochromis niloticus) Growth Hormone Receptor Type I

PubMed Central

2009-01-01

Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quantify specific genes. PMID:19495916

A Guideline to Family-Wide Comparative State-of-the-Art Quantitative RT-PCR Analysis Exemplified with a Brassicaceae Cross-Species Seed Germination Case Study[W][OA

PubMed Central

Graeber, Kai; Linkies, Ada; Wood, Andrew T.A.; Leubner-Metzger, Gerhard

2011-01-01

Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) data sets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability (i.e., low intersample transcript abundance variation) for correct target gene normalization. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue using tissue-specific transcriptome data sets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of Arabidopsis and Brassica napus microarray data sets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore, using germinating seeds, we exemplify optimization of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond Arabidopsis by establishing high-quality cross-species qRT-PCR. PMID:21666000

Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.

PubMed

Blanchard, Philippe; Regnault, Julie; Schurr, Frank; Dubois, Eric; Ribière, Magali

2012-03-01

Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee. Copyright © 2011 Elsevier B.V. All rights reserved.

Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula)

USDA-ARS?s Scientific Manuscript database

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference gene...

VARIATION OF THE EXPRESSION OF ENDOGENOUS "HOUSEKEEPING" GENES IN B[A]P TREATED MOUSE LUNGS MEASURED BY qRT-PCR

EPA Science Inventory

Quantitative RT-PCR is frequently used to analyze gene expression in different experimental systems. In this assay, housekeeping genes are frequently used to normalize for the variability between samples (relative quantification). We have examined the utility of using qRT-PCR and...

The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas)

USGS Publications Warehouse

Greenblatt, R.J.; Work, Thierry M.; Balazs, G.; Sutton, C.A.; Casey, R.N.; Casey, J.W.

2004-01-01

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles (Chelonia mydas) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches (Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

PubMed

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

Clinical Comparison of an Enhanced-Sensitivity Branched-DNA Assay and Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

PubMed Central

Nolte, Frederick S.; Boysza, Jodi; Thurmond, Cathy; Clark, W. Scott; Lennox, Jeffrey L.

1998-01-01

The performance characteristics of an enhanced-sensitivity branched-DNA assay (bDNA) (Quantiplex HIV-1 version 2.0; Chiron Corp., Emeryville, Calif.) and a reverse transcription (RT)-PCR assay (AMPLICOR HIV-1 Monitor; Roche Diagnostic Systems, Inc., Branchburg, N.J.) were compared in a molecular diagnostic laboratory. Samples used in this evaluation included linearity and reproducibility panels made by dilution of a human immunodeficiency virus type 1 (HIV-1) stock culture of known virus particle count in HIV-1-negative plasma, a subtype panel consisting of HIV-1 subtypes A through F at a standardized level, and 64 baseline plasma specimens from HIV-1-infected individuals. Plots of log10 HIV RNA copies per milliliter versus log10 nominal virus particles per milliliter demonstrated that both assays were linear over the stated dynamic ranges (bDNA, r = 0.98; RT-PCR, r = 0.99), but comparison of the slopes of the regression lines (bDNA, m = 0.96; RT-PCR, m = 0.83) suggested that RT-PCR had greater proportional systematic error. The between-run coefficients of variation for bDNA and RT-PCR were 24.3 and 34.3%, respectively, for a sample containing 1,650 nominal virus particles/ml and 44.0 and 42.7%, respectively, for a sample containing 165 nominal virus particles/ml. Subtypes B, C, and D were quantitated with similar efficiencies by bDNA and RT-PCR; however, RT-PCR was less efficient in quantitating subtypes A, E, and F. One non-B subtype was recognized in our clinical specimens based on the ratio of values obtained with the two methods. HIV-1 RNA was quantitated in 53 (83%) baseline plasma specimens by bDNA and in 55 (86%) specimens by RT-PCR. RT-PCR values were consistently greater than bDNA values, with population means of 142,419 and 67,580 copies/ml, respectively (P < 0.01). The results were highly correlated (r = 0.91), but the agreement was poor (mean difference in log10 copies per milliliter ± 2 standard deviations, 0.45 ± 0.61) for the 50 clinical specimens that gave discrete values with both methods. PMID:9508301

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.

PubMed

Clark-Langone, Kim M; Wu, Jenny Y; Sangli, Chithra; Chen, Angela; Snable, James L; Nguyen, Anhthu; Hackett, James R; Baker, Joffre; Yothers, Greg; Kim, Chungyeul; Cronin, Maureen T

2007-08-15

Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.

High-Throughput RT-PCR for small-molecule screening assays

PubMed Central

Bittker, Joshua A.

2012-01-01

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248

Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors.

PubMed

Bridge, Julia A

2017-01-01

The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription-polymerase chain reaction (RT-PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse-transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor-specific fusion gene events. Real-time RT-PCR (quantitative RT-PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre-analytic and analytic considerations of RT-PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11-19. © 2016 American Cancer Society. © 2016 American Cancer Society.

Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

PubMed

Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

2014-10-01

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

USGS Publications Warehouse

Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.

2006-01-01

The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.

Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil.

PubMed

Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

2014-07-01

The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

USDA-ARS?s Scientific Manuscript database

Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients.

PubMed

Kumar, Jyoti S; Saxena, Divyasha; Parida, Manmohan

2014-01-01

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

EPA Science Inventory

Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase.

PubMed

Marino-Merlo, Francesca; Frezza, Caterina; Papaianni, Emanuela; Valletta, Elena; Mastino, Antonio; Macchi, Beatrice

2017-11-01

Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.

Detection of growth hormone doping by gene expression profiling of peripheral blood.

PubMed

Mitchell, Christopher J; Nelson, Anne E; Cowley, Mark J; Kaplan, Warren; Stone, Glenn; Sutton, Selina K; Lau, Amie; Lee, Carol M Y; Ho, Ken K Y

2009-12-01

GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

PCR-free Quantification of Multiple Splice Variants in Cancer Gene by Surface Enhanced Raman Spectroscopy

PubMed Central

Sun, Lan; Irudayaraj, Joseph

2009-01-01

We demonstrate a surface enhanced Raman spectroscopy (SERS) based array platform to monitor gene expression in cancer cells in a multiplex and quantitative format without amplification steps. A strategy comprising of DNA/RNA hybridization, S1 nuclease digestion, and alkaline hydrolysis was adopted to obtain DNA targets specific to two splice junction variants Δ(9, 10) and Δ(5) of the breast cancer susceptibility gene 1 (BRCA1) from MCF-7 and MDA-MB-231 breast cancer cell lines. These two targets were identified simultaneously and their absolute quantities were estimated by a SERS strategy utilizing the inherent plasmon-phonon Raman mode of gold nanoparticle probes as a self-referencing standard to correct for variability in surface enhancement. Results were then validated by reverse transcription PCR (RT-PCR). Our proposed methodology could be expanded to a higher level of multiplexing for quantitative gene expression analysis of any gene without any amplification steps. PMID:19780515

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

PubMed

Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

2017-07-25

Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay.

PubMed

Ammour, Y; Faizuloev, E; Borisova, T; Nikonova, A; Dmitriev, G; Lobodanov, S; Zverev, V

2013-01-01

In this study, a rapid quantitative method using TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) has been developed for estimating the titers of measles, mumps and rubella (MMR) viruses in infected cell culture supernatants. The qPCR-RT assay was demonstrated to be a specific, sensitive, efficient and reproducible method. For MMR viral samples obtained during MMR viral propagations in Vero cells at a different multiplicity of infection, titers determined by the qPCR-RT assay have been compared with estimates of infectious virus obtained by a traditional commonly used method for MMR viruses - 50% cell culture infective dose (CCID(50)) assay, in paired samples. Pearson analysis evidenced a significant correlation between both methods for a certain period after viral inoculation. Furthermore, the established qPCR-RT assay was faster and less-laborious. The developed method could be used as an alternative method or a supplementary tool for the routine titer estimation during MMR vaccine production. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of four molecular assays for the detection of Tembusu virus.

PubMed

Tang, Yi; Yeh, Yin-Ting; Chen, Hao; Yu, Chunmei; Gao, Xuhui; Diao, Youxiang

2015-10-01

Tembusu virus (TMUV) belongs to the genus Flavivirus that may cause severe egg drop in ducks. In order to evaluate the most efficient TMUV detection method, the performances of a conventional RT-PCR (C-RT-PCR), a semi-nested PCR (SN-RT-PCR), a reverse-transcriptase real-time quantitative PCR (Q-RT-PCR), and a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) targeting the TMUV virus-specific NS5 gene were examined. In order to compare the sensitivity of these four techniques, two templates were used: (1) plasmid DNA that contained a partial region of the NS5 gene and (2) genomic RNA from TMUV-positive cell culture supernatants. The sensitivities using plasmid DNA detection by C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 2 × 10(4) copies/μL, 20 copies/μL, 2 copies/μL, and 20 copies/μL, respectively. The sensitivities using genomic RNA for the C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were 100 pg/tube, 100, 10, and 100 fg/tube, respectively. All evaluated assays were specific for TMUV detection. The TMUV-specific RNA was detected in cloacal swabs from experimentally infected ducks using these four methods with different rates (52-92%), but not in the control (non-inoculated) samples. The sensitivities of RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs collected from suspected TMUV infected ducks within 2 weeks of severe egg-drop were 38/69 (55.1%), 52/69 (75.4%), 57/69 (82.6%), and 55/69 (79.7%), respectively. In conclusion, both RT-LAMP and Q-RT-PCR can provide a rapid diagnosis of TMUV infection, but RT-LAMP is more useful in TMUV field situations or poorly equipped laboratories.

Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

PubMed

Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

2005-01-01

Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.

Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

PubMed

Valle-Maldonado, Marco I; Jácome-Galarza, Irvin E; Gutiérrez-Corona, Félix; Ramírez-Díaz, Martha I; Campos-García, Jesús; Meza-Carmen, Víctor

2015-03-01

Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish.

PubMed

Tattiyapong, P; Sirikanchana, K; Surachetpong, W

2018-02-01

Tilapia lake virus (TiLV) is an emerging pathogen associated with high mortalities of wild and farm-raised tilapia in different countries. In this study, a SYBR green-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting segment three of the virus was developed to detect and quantify TiLV in clinical samples and experimentally challenged fish. All 30 field samples with clinical signs and history consistent with TiLV infection were positive for TiLV as detected by the developed RT-qPCR method. The RT-qPCR technique provided 100 and 10,000 times more sensitive for virus detection than those offered by the RT-PCR and virus isolation in cell culture methods, respectively. The detection limit of the RT-qPCR method was as low as two viral copies/μl. Moreover, the RT-qPCR technique could be applied for TiLV detection in various fish tissues including gills, liver, brain, heart, anterior kidney and spleen. Significantly, this study delivered an accurate and reliable method for rapid detection of TiLV viruses that facilitates active surveillance programme and disease containment. © 2017 John Wiley & Sons Ltd.

Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2017-03-21

19-23]. Real-56 time reverse-transcription PCR remains the gold standard for quantitative , sensitive, and specific 57 detection of CCHFV; however...five-fold in two different series , and samples were run by real- time RT-PCR 116 in triplicate. The preliminary LOD was the lowest RNA dilution where...1 Sequence optimized real- time RT-PCR assay for detection of Crimean-Congo hemorrhagic fever 1 virus 2 3 JW Koehler1, KL Delp1, AT Hall1, SP

SUSD2 is frequently downregulated and functions as a tumor suppressor in RCC and lung cancer.

PubMed

Cheng, Yingying; Wang, Xiaolin; Wang, Pingzhang; Li, Ting; Hu, Fengzhan; Liu, Qiang; Yang, Fan; Wang, Jun; Xu, Tao; Han, Wenling

2016-07-01

Sushi domain containing 2 (SUSD2) is type I membrane protein containing domains inherent to adhesion molecules. There have been few reported studies on SUSD2, and they have mainly focused on breast cancer, colon cancer, and HeLa cells. However, the expression and function of SUSD2 in other cancers remain unclear. In the present study, we conducted an integrated bioinformatics analysis based on the array data from the GEO database and found a significant downregulation of SUSD2 in renal cell carcinoma (RCC) and lung cancer. Western blotting and quantitative RT-PCR (qRT-PCR) confirmed that SUSD2 was frequently decreased in RCC and lung cancer tissues compared with the corresponding levels in normal adjacent tissues. The restoration of SUSD2 expression inhibited the proliferation and clonogenicity of RCC and lung cancer cells, whereas the knockdown of SUSD2 promoted A549 cell growth. Our findings suggested that SUSD2 functions as a tumor suppressor gene (TSG) in RCC and lung cancer.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR.

PubMed

Krych, Łukasz; Kot, Witold; Bendtsen, Katja M B; Hansen, Axel K; Vogensen, Finn K; Nielsen, Dennis S

2018-01-01

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

PubMed

Mehle, Nataša; Dobnik, David; Ravnikar, Maja; Pompe Novak, Maruša

2018-05-03

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

Clinical evaluation of the BioFire FilmArray® BioThreat-E test for the diagnosis of Ebola Virus Disease in Guinea.

PubMed

Gay-Andrieu, Françoise; Magassouba, N'Fally; Picot, Valentina; Phillips, Cynthia L; Peyrefitte, Christophe N; Dacosta, Brigitte; Doré, Ahmadou; Kourouma, Fode; Ligeon-Ligeonnet, Véronique; Gauby, Corentin; Longuet, Christophe; Scullion, Matt; Faye, Ousmane; Machuron, Jean Louis; Miller, Mark

2017-07-01

The recent West Africa Ebola outbreak highlighted the need to provide access to rapid, safe and reliable Ebola Virus Disease diagnostics. The objective of this field study was to assess the clinical performance of the FilmArray ® BioThreat-E test for the detection of Ebola Zaïre virus in whole blood in symptomatic patients suspected of Ebola Virus Disease in Conakry (Guinea) from March to July 2015. The BioThreat-E test was compared to the two RT-PCRs, using serum, implemented at Donka Hospital in the emergency context: an in-house developed quantitative one-step RT-PCR adapted from the Weidmann technique, and the RealStar ® Filovirus RT-PCR Kit 1.0 (Altona-Diagnostics). We also assessed the performance of this assay in noninvasive specimens (urine and saliva) to detect infected patients. Of 135 patients enrolled and eligible for performance assessment on whole blood, the sensitivity was 95.7% [95% CI: 85.5-99.5] and specificity 100% [95% CI: 95.9-100]. Of the 37 symptomatic infected patients able to provide saliva and/or urine samples, 34 of the 35 saliva samples and all 3 of the urine samples were positive with the BioThreat-E test. This study showed that the FilmArray BioThreat-E test performs comparably to conventional molecular tests under field conditions, providing results and interpretation in approximately 1h. Due to its operational characteristics, it can be easily deployed in the field during an epidemic and could also be a useful tool for post-outbreak surveillance. Copyright © 2017 Elsevier B.V. All rights reserved.

One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan.

PubMed

Katayama, Hiroyuki; Haramoto, Eiji; Oguma, Kumiko; Yamashita, Hiromasa; Tajima, Atsushi; Nakajima, Hideichiro; Ohgaki, Shinichiro

2008-03-01

Sewerage systems are important nodes to monitor human enteric pathogens transmitted via water. A quantitative virus survey was performed once a month for a year to understand the seasonal profiles of noroviruses genotype 1 and genotype 2, enteroviruses, and adenoviruses in sewerage systems. A total of 72 samples of influent, secondary-treated wastewater before chlorination and effluent were collected from six wastewater treatment plants in Japan. Viruses were successfully recovered from 100ml of influent and 1000ml of the secondary-treated wastewater and effluent using the acid rinse method. Viruses were determined by the RT-PCR or PCR method to obtain the most probable number for each sample. All the samples were also assayed for fecal coliforms (FCs) by a double-layer method. The seasonal profiles of noroviruses genotype 1 and genotype 2 in influent were very similar, i.e. they were abundant in winter (from November to March) at a geometric mean value of 190 and 200 RT-PCR units/ml, respectively, and less frequent in summer (from June to September), at 4.9 and 9.1 RT-PCR units/ml, respectively. The concentrations of enteroviruses and adenoviruses were mostly constant all the year round, 17 RT-PCR units/ml and 320 PCR units/ml in influent, and 0.044 RT-PCR units/ml and 7.0 PCR units/ml in effluent, respectively.

Development and evaluation of ELISA and qRT-PCR for identification of Squash vein yellowing virus in cucurbits

USDA-ARS?s Scientific Manuscript database

Enzyme linked-immunosorbent assay (ELISA) and quantitative reverse transcription-PCR (qRT-PCR) assays were developed for identification of Squash vein yellowing virus (SqVYV), the cause of viral watermelon vine decline. Both assays were capable of detecting SqVYV in a wide range of cucurbit hosts. ...

Normalization of Reverse Transcription Quantitative PCR Data During Ageing in Distinct Cerebral Structures.

PubMed

Bruckert, G; Vivien, D; Docagne, F; Roussel, B D

2016-04-01

Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a routine method in many laboratories. Normalization of data from experimental conditions is critical for data processing and is usually achieved by the use of a single reference gene. Nevertheless, as pointed by the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, several reference genes should be used for reliable normalization. Ageing is a physiological process that results in a decline of many expressed genes. Reliable normalization of RT-qPCR data becomes crucial when studying ageing. Here, we propose a RT-qPCR study from four mouse brain regions (cortex, hippocampus, striatum and cerebellum) at different ages (from 8 weeks to 22 months) in which we studied the expression of nine commonly used reference genes. With the use of two different algorithms, we found that all brain structures need at least two genes for a good normalization step. We propose specific pairs of gene for efficient data normalization in the four brain regions studied. These results underline the importance of reliable reference genes for specific brain regions in ageing.

A microfluidic approach to parallelized transcriptional profiling of single cells.

PubMed

Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A; Brenner, David J; Lin, Qiao

2015-12-01

The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress.

Estrogen- and progesterone-receptor status in ECOG 2197: comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory.

PubMed

Badve, Sunil S; Baehner, Frederick L; Gray, Robert P; Childs, Barrett H; Maddala, Tara; Liu, Mei-Lan; Rowley, Steve C; Shak, Steven; Perez, Edith A; Perez, Edith D; Shulman, Lawrence J; Martino, Silvana; Davidson, Nancy E; Sledge, George W; Goldstein, Lori J; Sparano, Joseph A

2008-05-20

Central and local laboratory concordance for hormone receptor measurement is therapeutically important. This study compares estrogen receptor (ER) and progesterone receptor (PR) measured by local laboratory immunohistochemistry (IHC), central IHC, and central reverse-transcriptase polymerase chain reaction (RT-PCR) using a proprietary 21-gene assay. A case-control sample of 776 breast cancer patients from Eastern Cooperative Oncology Group (ECOG) study E2197 was evaluated. Central IHC Allred score for ER and PR was obtained using tissue microarrays and 1D5 ER antibody and 636 PR antibody. Quantitative RT-PCR for ER and PR in whole sections was performed using the 21-gene assay. For ER, the concordance between local and central IHC was 90% (95% CI, 88% to 92%), between local IHC and central RT-PCR was 91% (95% CI, 89% to 93%), and between central IHC and central RT-PCR was 93% (95% CI, 91% to 95%). For PR, the concordance between local IHC and central IHC was 84% (95% CI, 82% to 87%), between local IHC and central RT-PCR was 88% (95% CI, 85% to 90%), and between central IHC and central RT-PCR was 90% (95% CI, 88% to 92%). Although concordance was high, IHC ER-negative cases that were RT-PCR positive were more common than IHC ER-positive cases that were RT-PCR negative. In ER-positive patients, ER expression by central IHC Allred score was marginally associated with recurrence (P = .091), and ER expression by central RT-PCR was significantly associated with recurrence (P = .014). However, recurrence score, which incorporates additional genes/pathways, was a highly significant predictor of recurrence (P < .0001). There is a high degree of concordance among local IHC, central IHC, and central RT-PCR by the proprietary gene assay for ER and PR status. Although ER expression is marginally associated with relapse in ER-positive patients treated with chemohormonal therapy, recurrence score is a highly significant predictor of recurrence.

The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function.

PubMed

Khorsandi, Shirin Elizabeth; Quaglia, Alberto; Salehi, Siamak; Jassem, Wayel; Vilca-Melendez, Hector; Prachalias, Andreas; Srinivasan, Parthi; Heaton, Nigel

2015-01-01

Donation after cardiac death (DCD) livers are marginal organs for transplant and their use is associated with a higher risk of primary non function (PNF) or early graft dysfunction (EGD). The aim was to determine if microRNA (miRNA) was able to discriminate between DCD livers of varying clinical outcome. DCD groups were categorized as PNF retransplanted within a week (n=7), good functional outcome (n=7) peak aspartate transaminase (AST) ≤ 1000 IU/L and EGD (n=9) peak AST ≥ 2500 IU/L. miRNA was extracted from archival formalin fixed post-perfusion tru-cut liver biopsies. High throughput expression analysis was performed using miRNA arrays. Bioinformatics for expression data analysis was performed and validated with real time quantitative PCR (RT-qPCR). The function of miRNA of interest was investigated using computational biology prediction algorithms. From the array analysis 16 miRNAs were identified as significantly different (p<0.05). On RT-qPCR miR-155 and miR-940 had the highest expression across all three DCD clinical groups. Only one miRNA, miR-22, was validated with marginal significance, to have differential expression between the three groups (p=0.049). From computational biology miR-22 was predicted to affect signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. In conclusion, microRNA expression patterns have a low diagnostic potential clinically in discriminating DCD liver quality and outcome.

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements.

PubMed

Marini, N; Bevilacqua, C B; Büttow, M V; Raseira, M C B; Bonow, S

2017-05-25

Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

PubMed

Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

2015-10-24

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

Cloning of fox (Vulpes vulpes) Il2, Il6, Il10 and IFNgamma and analysis of their expression by quantitative RT-PCR in fox PBMC after in vitro stimulation by Concanavalin A.

PubMed

Rolland-Turner, Magali; Farré, Guillaume; Boué, Franck

2006-04-15

The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterised, and specific immunological tools are needed. A quantitative RT-PCR using SyBR Green to investigate fox cytokine expression after antigen PBMC in vitro re-stimulation is presented here. First, we cloned by homology with dog cytokine sequences the fox IL2, IL6, IL10, IFNgamma and a partial 18S sequence. Fox specific primers were then defined and used to set up a species-specific quantitative RT-PCR assay using SyBR Green and 18S housekeeping gene as internal standard. The technique was validated using total RNA from fox PBMC stimulated with a polyclonal activator, Concanavaline A.

Clinical comparison of branched DNA and reverse transcriptase-PCR and nucleic acid sequence-based amplification assay for the quantitation of circulating recombinant form_BC HIV-1 RNA in plasma.

PubMed

Pan, Pinliang; Tao, Xiaoxia; Zhang, Qi; Xing, Wenge; Sun, Xianguang; Pei, Lijian; Jiang, Yan

2007-12-01

To investigate the correlation between three viral load assays for circulating recombinant form (CRF)_BC. Recent studies in HIV-1 molecular epidemiology, reveals that CRF_BC is the dominant subtype of HIV-1 virus in mainland China, representing over 45% of the HIV-1 infected population. The performances of nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA) and reverse transcriptase polymerase chain reaction (RT-PCR) were compared for the HIV-1 viral load detection and quantitation of CRF_BC in China. Sixteen HIV-1 positive and three HIV-1 negative samples were collected. Sequencing of the positive samples in the gp41 region was conducted. The HIV-1 viral load values were determined using bDNA, RT-PCR and NASBA assays. Deming regression analysis with SPSS 12.0 (SPS Inc., Chicago, Illinois, USA) was performed for data analysis. Sequencing and phylogenetic analysis of env gene (gp41) region of the 16 HIV-1 positive clinical specimens from Guizhou Province in southwest China revealed the dominance of the subtype CRF_BC in that region. A good correlation of their viral load values was observed among three assays. Pearson's correlation between RT-PCR and bDNA is 0.969, Lg(VL)RT-PCR = 0.969 * Lg(VL)bDNA + 0.55; Pearson's correlation between RT-PCR and NASBA is 0.968, Lg(VL)RT-PCR = 0.968 * Lg(VL)NASBA + 0.937; Pearson's correlation between NASBA and bDNA is 0.980, Lg(VL)NASBA = 0.980 * Lg(VL)bDNA - 0.318. When testing with 3 different assays, RT-PCR, bDNA and NASBA, the group of 16 HIV-1 positive samples showed the viral load value was highest for RT-PCR, followed by bDNA then NASBA, which is consistent with the former results in subtype B. The three viral load assays are highly correlative for CRF_BC in China.

Does melatonin influence the apoptosis in rat uterus of animals exposed to continuous light?

PubMed

Ferreira, Cecília S; Carvalho, Kátia C; Maganhin, Carla C; Paiotti, Ana P R; Oshima, Celina T F; Simões, Manuel J; Baracat, Edmund C; Soares, José M

2016-02-01

Melatonin has been described as a protective agent against cell death and oxidative stress in different tissues, including in the reproductive system. However, the information on the action of this hormone in rat uterine apoptosis is low. Our objective was to evaluate the effects of melatonin on mechanisms of cell death in uterus of rats exposed to continuous light stress. Twenty adult Wistar rats were divided into two groups: GContr (vehicle control) and GExp which were treated with melatonin (0.4 mg/mL), both were exposed to continuous light for 90 days. The uterus was removed and processed for quantitative real time PCR (qRT-PCR), using PCR-array plates of the apoptosis pathway; for immunohistochemistry and TUNEL. The results of qRT-PCR of GEXP group showed up-regulation of 13 and 7, pro-apoptotic and anti-apoptotic genes, respectively, compared to GContr group. No difference in pro-apoptotic proteins (Bax, Fas and Faslg) expression was observed by immunohistochemistry, although the number of TUNEL-positive cells was lower in the group treated with melatonin compared to the group not treated with this hormone. Our data suggest that melatonin influences the mechanism and decreases the apoptosis in uterus of rats exposed to continuous light.

MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa.

PubMed

Calenda, Giulia; Villegas, Guillermo; Barnable, Patrick; Litterst, Claudia; Levendosky, Keith; Gettie, Agegnehu; Cooney, Michael L; Blanchard, James; Fernández-Romero, José A; Zydowsky, Thomas M; Teleshova, Natalia

2017-03-01

The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad-spectrum microbicide against HIV, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus in murine models. Recently, we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 coinfection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC, and compared quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Comparison of global brain gene expression profiles between inbred long-sleep and inbred short-sleep mice by high-density gene array hybridization.

PubMed

Xu, Y; Ehringer, M; Yang, F; Sikela, J M

2001-06-01

Inbred long-sleep (ILS) and short-sleep (ISS) mice show significant central nervous system-mediated differences in sleep time for sedative dose of ethanol and are frequently used as a rodent model for ethanol sensitivity. In this study, we have used complementary DNA (cDNA) array hybridization methodology to identify genes that are differentially expressed between the brains of ILS and ISS mice. To carry out this analysis, we used both the gene discovery array (GDA) and the Mouse GEM 1 Microarray. GDA consists of 18,378 nonredundant mouse cDNA clones on a single nylon filter. Complex probes were prepared from total brain mRNA of ILS or ISS mice by using reverse transcription and 33P labeling. The labeled probes were hybridized in parallel to the gene array filters. Data from GDA experiments were analyzed with SQL-Plus and Oracle 8. The GEM microarray includes 8,730 sequence-verified clones on a glass chip. Two fluorescently labeled probes were used to hybridize a microarray simultaneously. Data from GEM experiments were analyzed by using the GEMTools software package (Incyte). Differentially expressed genes identified from each method were confirmed by relative quantitative reverse transcription-polymerase chain reaction (RT-PCR). A total of 41 genes or expressed sequence tags (ESTs) display significant expression level differences between brains of ILS and ISS mice after GDA, GEM1 hybridization, and quantitative RT-PCR confirmation. Among them, 18 clones were expressed higher in ILS mice, and 23 clones were expressed higher in ISS mice. The individual gene or EST's function and mapping information have been analyzed. This study identified 41 genes that are differentially expressed between brains of ILS and ISS mice. Some of them may have biological relevance in mediation of phenotypic variation between ILS and ISS mice for ethanol sensitivity. This study also demonstrates that parallel gene expression comparison with high-density cDNA arrays is a rapid and efficient way to discover potential genes and pathways involved in alcoholism and alcohol-related physiologic processes.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus.

PubMed

Hui, Yuan; Wu, Zhiming; Qin, Zhiran; Zhu, Li; Liang, Junhe; Li, Xujuan; Fu, Hanmin; Feng, Shiyu; Yu, Jianhai; He, Xiaoen; Lu, Weizhi; Xiao, Weiwei; Wu, Qinghua; Zhang, Bao; Zhao, Wei

2018-06-01

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold (Ct) value was linear from 10 1 to 10 8  copy/μL, with a standard curve R 2 of 0.999 and amplification efficiency of 92.203%; however, a concentration as low as 1 copy/μL could not be detected. In comparison with RT-qPCR, the ddPCR method resulted in a linear range of 10 1 -10 4  copy/μL and was able to detect concentrations as low as 1 copy/μL. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples (above 10 1  copy/μL), while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Gene expression profile of steroid-induced necrosis of femoral head of rats.

PubMed

Tong, Peijian; Wu, Chengliang; Jin, Hongting; Mao, Qiang; Yu, Nanze; Holz, Jonathan D; Shan, Letian; Liu, Hui; Xiao, Luwei

2011-10-01

The key to treating steroid-induced necrosis of femoral heads (SINFH) is early diagnosis. Dramatic improvements in diagnosis could be made if the pathogenesis of SINFH was more fully understood; however, the underlying mechanism of this disease is currently unknown. To explore the potential mechanism of SINFH, we performed gene array analysis on a rat model of the disease and compare the expression profile with that of normal rats. A quantitative RT-PCR and immunohistochemistry (IHC) assays were used to confirm the microarray results. Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (deposited in GenBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and transcription related genes. The results of quantitative RT-PCR and IHC were consistent with gene chip results. Our findings indicate that many genes involved in diverse signaling pathways were differentially expressed between SINFH rats and normal rats. Furthermore, our findings suggest that the development of SINFH is a complicated and dynamic process affected by multiple factors and signaling pathways and regulated by various genes.

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

PubMed

Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

2017-09-01

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.

PubMed

Niu, Longjian; Tao, Yan-Bin; Chen, Mao-Sheng; Fu, Qiantang; Li, Chaoqiong; Dong, Yuling; Wang, Xiulan; He, Huiying; Xu, Zeng-Fu

2015-06-03

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

Performance Evaluation of the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit: Comparison with the Roche COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test Ver.2.0 for Quantification of HIV-1 Viral Load in Indonesia.

PubMed

Kosasih, Agus Susanto; Sugiarto, Christine; Hayuanta, Hubertus Hosti; Juhaendi, Runingsih; Setiawan, Lyana

2017-08-08

Measurement of viral load in human immunodeficiency virus type 1 (HIV-1) infected patients is essential for the establishment of a therapeutic strategy. Several assays based on qPCR are available for the measurement of viral load; they differ in sample volume, technology applied, target gene, sensitivity and dynamic range. The Bioneer AccuPower® HIV-1 Quantitative RT-PCR is a novel commercial kit that has not been evaluated for its performance. This study aimed to evaluate the performance of the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit. In total, 288 EDTA plasma samples from the Dharmais Cancer Hospital were analyzed with the Bioneer AccuPower® HIV-1 Quantitative RT-PCR kit and the Roche COBAS? AmpliPrep/COBAS® TaqMan® HIV-1 version 2.0 (CAP/CTM v2.0). The performance of the Bioneer assay was then evaluated against the Roche CAP/CTM v2.0. Overall, there was good agreement between the two assays. The Bioneer assay showed significant linear correlation with CAP/CTM v2.0 (R2=0.963, p<0.001) for all samples (N=118) which were quantified by both assays, with high agreement (94.9%, 112/118) according to the Bland-Altman model. The mean difference between the quantitative values measured by Bioneer assay and CAP/CTM v2.0 was 0.11 Log10 IU/mL (SD=0.26). Based on these results, the Bioneer assay can be used to quantify HIV-1 RNA in clinical laboratories.

Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

PubMed Central

Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

2015-01-01

To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

PubMed Central

Fout, G. Shay; Cashdollar, Jennifer L.; Griffin, Shannon M.; Brinkman, Nichole E.; Varughese, Eunice A.; Parshionikar, Sandhya U.

2016-01-01

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water. PMID:26862985

Application of a Master Equation for Quantitative mRNA Analysis Using qRT-PCR

USDA-ARS?s Scientific Manuscript database

The qRT-PCR has been widely accepted as the assay of choice for mRNA quantification. Gene expression as measured by mRNA dynamics varies in response to different conditions and environmental stimuli. For conventional practice, housekeeping genes have been applied as internal reference for data nor...

Enzymatic and viability RT-qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment.

PubMed

Monteiro, S; Santos, R

2018-04-01

To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses. © 2017 The Society for Applied Microbiology.

Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains.

PubMed

Nieser, Maike; Henopp, Tobias; Brix, Joachim; Stoß, Laura; Sitek, Barbara; Naboulsi, Wael; Anlauf, Martin; Schlitter, Anna M; Klöppel, Günter; Gress, Thomas; Moll, Roland; Bartsch, Detlef K; Heverhagen, Anna E; Knoefel, Wolfram T; Kaemmerer, Daniel; Haybaeck, Johannes; Fend, Falko; Sperveslage, Jan; Sipos, Bence

2017-01-01

Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18. © 2016 S. Karger AG, Basel.

Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Schonfeld, Julie; Tran, Luan

2016-01-01

The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis in Isatis indigotica Fort.

PubMed Central

Li, Tao; Wang, Jing; Lu, Miao; Zhang, Tianyi; Qu, Xinyun; Wang, Zhezhi

2017-01-01

Due to its sensitivity and specificity, real-time quantitative PCR (qRT-PCR) is a popular technique for investigating gene expression levels in plants. Based on the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines, it is necessary to select and validate putative appropriate reference genes for qRT-PCR normalization. In the current study, three algorithms, geNorm, NormFinder, and BestKeeper, were applied to assess the expression stability of 10 candidate reference genes across five different tissues and three different abiotic stresses in Isatis indigotica Fort. Additionally, the IiYUC6 gene associated with IAA biosynthesis was applied to validate the candidate reference genes. The analysis results of the geNorm, NormFinder, and BestKeeper algorithms indicated certain differences for the different sample sets and different experiment conditions. Considering all of the algorithms, PP2A-4 and TUB4 were recommended as the most stable reference genes for total and different tissue samples, respectively. Moreover, RPL15 and PP2A-4 were considered to be the most suitable reference genes for abiotic stress treatments. The obtained experimental results might contribute to improved accuracy and credibility for the expression levels of target genes by qRT-PCR normalization in I. indigotica. PMID:28702046

Design and optimization of reverse-transcription quantitative PCR experiments.

PubMed

Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

2009-10-01

Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

Nucleic Acid Research Group (NARG) 2009-2010 Study : Optimal Priming Strategies for cDNA Synthesis in Real-Time RT-qPCR

PubMed Central

Hunter, T.C.; Knudtson, K.L.; Nadella, V.; Sol-Church, K.; Taylor, W.L.; Tighe, S.; Yueng, A.T.; Chittur, S.

2010-01-01

r1-1 Real-time reverse transcriptase quantitative PCR (RT-qPCR) is a widely used technique for measuring transcript levels. Priming strategy and reverse transcriptase enzyme are key elements that affect sensitivity and variability of RT-qPCR and microarray results. Previously, the Nucleic Acid Research Group (NARG) had conducted preliminary studies within the group to examine the effects of priming strategy on generating cDNA for use with qPCR. This year's study was an open study in which the qPCR community was invited to participate. Participants received the RT primers and RNA template and were asked to perform the RT reaction using their preferred reaction conditions. Each participating laboratory was provided at least two RNA templates of varying quality. The RT products were returned to the NARG and all RT reactions were used in a qPCR reaction. The qPCR assays looked at three genes of varying abundance, b-actin (high copy), b-glucuronidase (medium copy) and TATA binding protein (low copy) as well as varying distance from the 3? end for each transcript. Results from participating laboratories will be evaluated to determine the impact of priming strategy, assay chemistry and experimental setup on the RT step. Additionally, we will address the impact of RNA integrity on cDNA synthesis.

Development and Application of Quantitative Detection Method for Viral Hemorrhagic Septicemia Virus (VHSV) Genogroup IVa

PubMed Central

Kim, Jong-Oh; Kim, Wi-Sik; Kim, Si-Woo; Han, Hyun-Ja; Kim, Jin Woo; Park, Myoung Ae; Oh, Myung-Joo

2014-01-01

Viral hemorrhagic septicemia virus (VHSV) is a problematic pathogen in olive flounder (Paralichthys olivaceus) aquaculture farms in Korea. Thus, it is necessary to develop a rapid and accurate diagnostic method to detect this virus. We developed a quantitative RT-PCR (qRT-PCR) method based on the nucleocapsid (N) gene sequence of Korean VHSV isolate (Genogroup IVa). The slope and R2 values of the primer set developed in this study were −0.2928 (96% efficiency) and 0.9979, respectively. Its comparison with viral infectivity calculated by traditional quantifying method (TCID50) showed a similar pattern of kinetic changes in vitro and in vivo. The qRT-PCR method reduced detection time compared to that of TCID50, making it a very useful tool for VHSV diagnosis. PMID:24859343

Detection of doublecortin domain-containing 2 (DCDC2), a new candidate tumor suppressor gene of hepatocellular carcinoma, by triple combination array analysis

PubMed Central

2013-01-01

Background To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. Methods A surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2. Results DCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0–1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048). Conclusions The present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC. PMID:24034596

Evaluation of changes in periodontal bacteria in healthy dogs over 6 months using quantitative real-time PCR.

PubMed

Maruyama, N; Mori, A; Shono, S; Oda, H; Sako, T

2018-03-01

Porphyromonas gulae, Tannerella forsythia and Campylobacter rectus are considered dominant periodontal pathogens in dogs. Recently, quantitative real-time PCR (qRT-PCR) methods have been used for absolute quantitative determination of oral bacterial counts. The purpose of the present study was to establish a standardized qRT-PCR procedure to quantify bacterial counts of the three target periodontal bacteria (P. gulae, T. forsythia and C. rectus). Copy numbers of the three target periodontal bacteria were evaluated in 26 healthy dogs. Then, changes in bacterial counts of the three target periodontal bacteria were evaluated for 24 weeks in 7 healthy dogs after periodontal scaling. Analytical evaluation of each self-designed primer indicated acceptable analytical imprecision. All 26 healthy dogs were found to be positive for P. gulae, T. forsythia and C. rectus. Median total bacterial counts (copies/ng) of each target genes were 385.612 for P. gulae, 25.109 for T. forsythia and 5.771 for C. rectus. Significant differences were observed between the copy numbers of the three target periodontal bacteria. Periodontal scaling reduced median copy numbers of the three target periodontal bacteria in 7 healthy dogs. However, after periodontal scaling, copy numbers of all three periodontal bacteria significantly increased over time (p<0.05, Kruskal-Wallis test) (24 weeks). In conclusion, our results demonstrated that qRT-PCR can accurately measure periodontal bacteria in dogs. Furthermore, the present study has revealed that qRT-PCR method can be considered as a new objective evaluation system for canine periodontal disease. Copyright© by the Polish Academy of Sciences.

Evaluation and selection of internal reference genes from two- and six-row U.S. malting barley varieties throughout micromalting for use in RT-qPCR

USDA-ARS?s Scientific Manuscript database

Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR) is a popular method for measuring transcript abundance. The most commonly used method of interpretation is relative quantification and thus necessitates the use of normalization controls (i.e. reference genes) to standardize tran...

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Development of a quantitative PCR for detection of Lactobacillus plantarum starters during wine malolactic fermentation.

PubMed

Cho, Gyu-Sung; Krauss, Sabrina; Huch, Melanie; Du Toit, Maret; Franz, Charles M A P

2011-12-01

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at 3.6 × 10(6) CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above 10(5)/ml for approx. 10 days, after which cell numbers decreased to levels of 10(3) CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. 1 × 10(2) CFU/ml was detected. The minimum detection level for quantitative PCR in this study was 1 × 10(2) to 1 × 10(3) CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

A candidate pheromone receptor and two odorant receptors of the hawkmoth Manduca sexta.

PubMed

Patch, Harland M; Velarde, Rodrigo A; Walden, Kimberly K O; Robertson, Hugh M

2009-05-01

In this study, we cloned and characterized three Manduca sexta odorant receptors (ORs). One receptor is a putative pheromone receptor expressed exclusively in a cell associated with male-specific type-I trichoid sensilla. We describe the results of real-time PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) experiments that show MsextaOR1 is expressed only in male antennae. In situ hybridization labels a single cell associated with type-1 trichoid sensilla, which houses two neurons that have been previously determined to respond to the major components of the pheromone blend. The second receptor, MsextaOR2, was discovered using degenerate primers designed to conserved motifs of a unique group ORs that share as much as 88% identity. Comparison of RT-PCR, qRT-PCR, and in situ hybridization results with those of ORs in the Drosophila melanogaster Or83b subfamily shows a strong sequence and expression pattern similarity. The third receptor, MsextaOR3, was found by 5'-end sequencing of a normalized and subtracted cDNA library from male M. sexta antennae. RT-PCR and qRT-PCR show that this receptor is expressed only in male and female antennae. These are the first ORs, including a putative pheromone receptor, to be described from M. sexta.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR.

PubMed

Stein, Erica V; Duewer, David L; Farkas, Natalia; Romsos, Erica L; Wang, Lili; Cole, Kenneth D

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Steps to achieve quantitative measurements of microRNA using two step droplet digital PCR

PubMed Central

Duewer, David L.; Farkas, Natalia; Romsos, Erica L.; Wang, Lili; Cole, Kenneth D.

2017-01-01

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values. PMID:29145448

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

PubMed

Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L

2017-08-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

PubMed Central

Hout, David R.; Lawrence, Kasey; Morris, Stephan W.; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly

2017-01-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK. PMID:28763012

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

PubMed Central

2012-01-01

Background Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. Methods Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients. Results We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian. Conclusions Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis. PMID:22691236

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

PubMed

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

A novel quantitative reverse-transcription PCR (qRT-PCR) for the enumeration of total bacteria, using meat micro-flora as a model.

PubMed

Dolan, Anthony; Burgess, Catherine M; Barry, Thomas B; Fanning, Seamus; Duffy, Geraldine

2009-04-01

A sensitive quantitative reverse-transcription PCR (qRT-PCR) method was developed for enumeration of total bacteria. Using two sets of primers separately to target the ribonuclease-P (RNase P) RNA transcripts of gram positive and gram negative bacteria. Standard curves were generated using SYBR Green I kits for the LightCycler 2.0 instrument (Roche Diagnostics) to allow quantification of mixed microflora in liquid media. RNA standards were used and extracted from known cell equivalents and subsequently converted to cDNA for the construction of standard curves. The number of mixed bacteria in culture was determined by qRT-PCR, and the results correlated (r(2)=0.88, rsd=0.466) with the total viable count over the range from approx. Log(10) 3 to approx. Log(10) 7 CFU ml(-1). The rapid nature of this assay (8 h) and its potential as an alternative method to the standard plate count method to predict total viable counts and shelf life are discussed.

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

USGS Publications Warehouse

Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

2002-01-01

A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.

Comparative analysis of gene expression level by quantitative real-time PCR has limited application in objects with different morphology.

PubMed

Demidenko, Natalia V; Penin, Aleksey A

2012-01-01

qRT-PCR is a generally acknowledged method for gene expression analysis due to its precision and reproducibility. However, it is well known that the accuracy of qRT-PCR data varies greatly depending on the experimental design and data analysis. Recently, a set of guidelines has been proposed that aims to improve the reliability of qRT-PCR. However, there are additional factors that have not been taken into consideration in these guidelines that can seriously affect the data obtained using this method. In this study, we report the influence that object morphology can have on qRT-PCR data. We have used a number of Arabidopsis thaliana mutants with altered floral morphology as models for this study. These mutants have been well characterised (including in terms of gene expression levels and patterns) by other techniques. This allows us to compare the results from the qRT-PCR with the results inferred from other methods. We demonstrate that the comparison of gene expression levels in objects that differ greatly in their morphology can lead to erroneous results.

Trends and advances in food analysis by real-time polymerase chain reaction.

PubMed

Salihah, Nur Thaqifah; Hossain, Mohammad Mosharraf; Lubis, Hamadah; Ahmed, Minhaz Uddin

2016-05-01

Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.

Multidisciplinary Biomarkers of Early Mammary Carcinogenesis

DTIC Science & Technology

2011-04-01

cDNA prepared. Quantitative real-time PCR (qRT-PCR) was then performed on the cDNA. All qRT-PCR reactions were performed in triplicate. ESR1 ...in Figure 4, all ER(+) cells express ESR1 at high levels (at least 4 fold higher than ER(-) cell lines). A Pearson correlation coefficient was...calculated to determine the linear relationship between the optical redox ratio and ESR1 expression levels and found to be significant (p = 0.0024, r

Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

USDA-ARS?s Scientific Manuscript database

Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

PubMed

Thakore, Pratiksha I; Gersbach, Charles A

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

PubMed

Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

2016-01-01

Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

PubMed Central

Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

2011-01-01

Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

PubMed Central

Niu, Longjian; Tao, Yan-Bin; Chen, Mao-Sheng; Fu, Qiantang; Li, Chaoqiong; Dong, Yuling; Wang, Xiulan; He, Huiying; Xu, Zeng-Fu

2015-01-01

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi. PMID:26047338

Promoter methylation assay of SASH1 gene in hepatocellular carcinoma.

PubMed

Peng, Liu; Wei, He; Liren, Li

2014-01-01

To analyse the relationship between the expression of SASH1 and its methylation level in human hepatocellular carcinoma. Expression levels of SASH1 were examined with real-time PCR (RT-PCR) in tissues and cells, and methylation analysis was performed with MassArray. The expression levels of SASH1 were strongly reduced in liver cancer tissues compared with adjacent normal tissues. Quantitative methylation analysis by MassArray revealed different CpG sites in SASH1 promoter shared similar methylation pattern between liver cancer tissues and adjacent normal tissues and the CpG sites of significant difference in methylation level were found as follows: CpG_3, CpG_17, CpG_21.22, CpG_25, CpG_26.27, CpG_28, CpG_34.35.36 and CpG_51.52. Moreover, 5-aza-2'-deoxycytidine treatment of Hep-G2 cell line caused significant elevation of SASH1 mRNA. Based on these data, we propose that increase of DNA methylation degree in the promoter region of SASH1 gene, particularly CpG_26.27 sites, possibly repressed SASH1 expression in liver cancer.

Promoter methylation assay of SASH1 gene in breast cancer.

PubMed

Sheyu, Lin; Hui, Liu; Junyu, Zhang; Jiawei, Xu; Honglian, Wang; Qing, Sang; Hengwei, Zhang; Xuhui, Guo; Qinghe, Xing; Lin, He

2013-01-01

To analyze the relationship between the expression of SASH1 and its methylation level of SASH1 gene promoter in human breast cancer. Expression levels of SASH1 were examined in breast cancer tissues and adjacent normal tissues with immunohistochemistry and with real time PCR (RT-PCR) methylation analysis was performed with MassArray. Immunohistochemistry showed that SASH1 expression was strongly reduced in breast cancer compared with adjacent normal tissues. Quantitative methylation analysis by MassArray revealed that CpG sites in SASH1 promoter shared similar methylation pattern in tumor tissue and adjacent normal tissue. The CpG sites with significant difference in methylation level were CpG_26.27 and CpG_54.55. Moreover, 5-aza-2'-deoxycytidine (5-Aza-dc) treatment of tumor cell line MDA-MB-231 caused significant elevation of SASH1 mRNA. Based on these data, we propose that increase of DNA methylation level in the promoter region of gene SASH1, particularly CpG_26.27 or CpG_54.55 sites, possibly repressed SASH1 expression in breast cancer.

Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

PubMed

Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

2018-01-01

A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

PubMed

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

PubMed

De Luca, Eliana; Crisi, Paolo Emidio; Di Domenico, Marco; Malatesta, Daniela; Vincifori, Giacomo; Di Tommaso, Morena; Di Guardo, Giovanni; Di Francesco, Gabriella; Petrini, Antonio; Savini, Giovanni; Boari, Andrea; Lorusso, Alessio

2018-05-03

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis.

PubMed

Rocha, Danilo J P; Santos, Carolina S; Pacheco, Luis G C

2015-09-01

The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.

The use of quantitative real-time reverse transcriptase PCR for 5' and 3' portions of ALK transcripts to detect ALK rearrangements in lung cancers.

PubMed

Wang, Rui; Pan, Yunjian; Li, Chenguang; Hu, Haichuan; Zhang, Yang; Li, Hang; Luo, Xiaoyang; Zhang, Jie; Fang, Zhaoyuan; Li, Yuan; Shen, Lei; Ji, Hongbin; Garfield, David; Sun, Yihua; Chen, Haiquan

2012-09-01

Approximately 3% to 7% of non-small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5' and 3' portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5' and 3' portions of ALK mRNA. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5' and 3' portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5' RACE coupling sequencing identified the fusion variants. Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20). Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5' RACE to this method should further facilitate rapid identification of novel ALK fusion genes. ©2012 AACR.

Validation of the Pockit Dengue Virus Reagent Set for Rapid Detection of Dengue Virus in Human Serum on a Field-Deployable PCR System.

PubMed

Tsai, Jih-Jin; Liu, Li-Teh; Lin, Ping-Chang; Tsai, Ching-Yi; Chou, Pin-Hsing; Tsai, Yun-Long; Chang, Hsiao-Fen Grace; Lee, Pei-Yu Alison

2018-05-01

Dengue virus (DENV) infection, a mosquito-borne disease, is a major public health problem in tropical countries. Point-of-care DENV detection with good sensitivity and specificity enables timely early diagnosis of DENV infection, facilitating effective disease management and control, particularly in regions of low resources. The Pockit dengue virus reagent set (GeneReach Biotech), a reverse transcription insulated isothermal PCR (RT-iiPCR), is available to detect all four serotypes of DENV on the field-deployable Pockit system, which is ready for on-site applications. In this study, analytical and clinical performances of the assay were evaluated. The index assay did not react with 14 non-DENV human viruses, indicating good specificity. Compared to the U.S. CDC DENV-1-4 real-time quantitative RT-PCR (qRT-PCR) assay, testing with serial dilutions of virus-spiked human sera demonstrated that the index assay had detection endpoints that were separately comparable with the 4 serotypes. Excellent reproducibility was observed among repeat tests done by six operators at three sites. In clinical performance, 195 clinical sera collected around Kaohsiung city in 2012 and 21 DENV-4-spiked sera were tested with the RT-iiPCR and qRT-PCR assays in parallel. The 121 (11 DENV-1, 78 DENV-2, 11 DENV-3, and 21 DENV-4) qRT-PCR-positive and 95 qRT-PCR-negative samples were all positive and negative by the RT-iiPCR reagent results, respectively, demonstrating high (100%) interrater agreement (95% confidence interval [CI 95% ], ∼98.81% to 100%; κ = 1). With analytical and clinical performance equivalent to those of the reference qRT-PCR assay, the index PCR assay on the field-deployable system can serve as a highly sensitive and specific on-site tool for DENV detection. Copyright © 2018 American Society for Microbiology.

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Targeting neuroendocrine differentiation for prostate cancer radiosensitization

DTIC Science & Technology

2017-12-01

Secondary HRP-conjugated antibodies were purchased fromGEHealthcare UK Ltd. (Buckinghamshire, UK). 2.5. RNA isolation and quantitative real- time PCR (qRT...gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T))Method,Methods 25 (2001) 402–408. [47] T.K. Kelly, T.B. Miranda, G...relative gene expression data using real- time quantitative PCR and the 2(−Delta Delta C(T)) method, Methods 25 (2001) 402–408. [37] J. Ren, L. Wen, X

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

USDA-ARS?s Scientific Manuscript database

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

Human epidermal growth factor receptor 2 assessment in a case-control study: comparison of fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction performed by central laboratories.

PubMed

Baehner, Frederick L; Achacoso, Ninah; Maddala, Tara; Shak, Steve; Quesenberry, Charles P; Goldstein, Lynn C; Gown, Allen M; Habel, Laurel A

2010-10-01

The optimal method to assess human epidermal growth factor receptor 2 (HER2) status remains highly controversial. Before reporting patient HER2 results, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines mandate that laboratories demonstrate ≥ 95% concordance to another approved laboratory or methodology. Here, we compare central laboratory HER2 assessed by fluorescence in situ hybridization (FISH) and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using Oncotype DX in lymph node-negative, chemotherapy-untreated patients from a large Kaiser Permanente case-control study. Breast cancer specimens from the Kaiser-Genomic Health study were examined. Central FISH assessment of HER2 amplification and polysomy 17 was conducted by PhenoPath Laboratories (ratios > 2.2, 1.8 to 2.2, and < 1.8 define HER2 positive, HER2 equivocal, and HER2 negative, respectively). HER2 expression by RT-PCR was conducted using Oncotype DX by Genomic Health (normalized expression units ≥ 11.5, 10.7 to < 11.5, and < 10.7 define HER2 positive, HER2 equivocal, and HER2 negative, respectively). Concordance analyses followed ASCO/CAP guidelines. HER2 concordance by central FISH and central RT-PCR was 97% (95% CI, 96% to 99%). Twelve percent (67 of 568 patients) and 11% (60 of 568 patients) of patients were HER2 positive by RT-PCR and FISH, respectively. HER2-positive patients had increased odds of dying from breast cancer compared with HER2-negative patients. Polysomy 17 was demonstrated in 12.5% of all patients and 33% of FISH-positive patients. Nineteen of 20 FISH-positive patients with polysomy 17 were also RT-PCR HER2 positive. Although not statistically significantly different, HER2-positive/polysomy 17 patients tended to have the worst prognosis, followed by HER2-positive/eusomic, HER2-negative/polysomy 17, and HER2-negative/eusomic patients. There is a high degree of concordance between central FISH and quantitative RT-PCR using Oncotype DX for HER2 status, and the assay warrants additional study in a trastuzumab-treated population.

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis.

PubMed

Nguyen, Duc Quan; Eamens, Andrew L; Grof, Christopher P L

2018-01-01

Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail ( Setaria viridis ) has recently been proposed as a potential experimental model for the study of C 4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens -mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis ( S. viridis ). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data. Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ), 5 '- ADENYLYLSULFATE REDUCTASE 6 ( ASPR6 ) and DUAL SPECIFICITY PHOSPHATASE ( DUSP ) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A , ASPR6 and DUSP , were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE ( CAD ) genes across the same tissues. This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C 4 crop species.

Altered serum microRNAs as biomarkers for the early diagnosis of pulmonary tuberculosis infection

PubMed Central

2012-01-01

Background Pulmonary tuberculosis (TB) is a highly lethal infectious disease and early diagnosis of TB is critical for the control of disease progression. The objective of this study was to profile a panel of serum microRNAs (miRNAs) as potential biomarkers for the early diagnosis of pulmonary TB infection. Methods Using TaqMan Low-Density Array (TLDA) analysis followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation, expression levels of miRNAs in serum samples from 30 patients with active tuberculosis and 60 patients with Bordetella pertussis (BP), varicella-zoster virus (VZV) and enterovirus (EV) were analyzed. Results The Low-Density Array data showed that 97 miRNAs were differentially expressed in pulmonary TB patient sera compared with healthy controls (90 up-regulated and 7 down-regulated). Following qRT-PCR confirmation and receiver operational curve (ROC) analysis, three miRNAs (miR-361-5p, miR-889 and miR-576-3p) were shown to distinguish TB infected patients from healthy controls and other microbial infections with moderate sensitivity and specificity (area under curve (AUC) value range, 0.711-0.848). Multiple logistic regression analysis of a combination of these three miRNAs showed an enhanced ability to discriminate between these two groups with an AUC value of 0.863. Conclusions Our study suggests that altered levels of serum miRNAs have great potential to serve as non-invasive biomarkers for early detection of pulmonary TB infection. PMID:23272999

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

PubMed

Die, Jose V; Baldwin, Ransom L; Rowland, Lisa J; Li, Robert; Oh, Sunghee; Li, Congjun; Connor, Erin E; Ranilla, Maria-Jose

2017-01-01

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

PubMed

Piller, Nicolas; Decosterd, Isabelle; Suter, Marc R

2013-07-10

The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

PubMed

Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

2015-07-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs

PubMed Central

SEHATA, Go; SATO, Hiroaki; ITO, Toshihiro; IMAIZUMI, Yoshitaka; NORO, Taichi; OISHI, Eiji

2015-01-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

PubMed

Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé

2014-01-01

In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.

PubMed

Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin

2014-01-01

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.

PubMed

Sandstedt, Mikael; Jonsson, Marianne; Asp, Julia; Dellgren, Göran; Lindahl, Anders; Jeppsson, Anders; Sandstedt, Joakim

2015-12-01

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.

Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissue.

PubMed

Daniele, Lorenzo; Annaratone, Laura; Allia, Elena; Mariani, Sara; Armando, Enrico; Bosco, Martino; Macrì, Luigia; Cassoni, Paola; D'Armento, Giuseppe; Bussolati, Gianni; Cserni, Gabor; Sapino, Anna

2009-09-01

The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT-PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step-sectioning protocol at 100 micron-intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT-PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT-PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT-PCR negative cases, metastases were detected only on sections distant from those analysed by RT-PCR. CEA and/or CK19 were positive by RT-PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.

[Quantitative real-time PCR for target periodontal bacteria in subgingival plaque before and after local delivery of periocline, scaling and root planning].

PubMed

Deng, Shu-li; Wang, Ying; He, Jia-yan; Chen, Zhuo; Chen, Hui

2013-06-01

To compare the copy number of Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi) in subgingival plaque before and after local delivery of periocline (2% minocycline hydrochloride ointment, MO), scaling and root planning (SRP) by quantitative real-time PCR (qRT-PCR) and evaluate the efficacy. Sixty-two adults with moderate to severe chronic periodontitis were selected in the study. Microbial samples were taken from pocket before and after MO and SRP(7d). The samples were evaluated by qRT-PCR for Pg and Pi. Microbiological effectiveness of treatments was assessed using Kruskal-Wallis and Wilcoxon rank-sum test. All tests were two-sided with a significance level of 0.05. All analyses were conducted with SAS 9.1.3 software package. The copy number of Pg and Pi in subgingival plaque was 10(3)-10(6) and 10(2)-10(6). Bacterial loads of Pg were reduced in SPR+ MO, SRP and MO site. The counts of Pi decreased in SRP+ MO sites compared with those in the MO or SRP alone sites significantly (P<0.05). Quantitative real-time PCR (qRT-PCR) is used as a powerful tool with high sensitivity and specificity to quantitatively assess target periodontal bacteria. The results show that subgingival administration of MO and SRP was effective for reducing pathogenic bacteria and improving clinical outcome. Supported by 2011 National Clinical Specialist Construction Project; Natural Science Foundation of Zhejiang Province(LY13H140002); Education Department Funds of Zhejiang Province(20061258) and Medical General Research Project of Zhejiang Province(2012KYB121).

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014.

PubMed

Zhuge, Jian; Vail, Eric; Bush, Jeffrey L; Singelakis, Lauren; Huang, Weihua; Nolan, Sheila M; Haas, Janet P; Engel, Helen; Della Posta, Millicent; Yoon, Esther C; Fallon, John T; Wang, Guiqing

2015-06-01

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5' untranslated region (5'-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of a Real-Time Reverse Transcription-PCR Assay for Detection of Enterovirus D68 in Clinical Samples from an Outbreak in New York State in 2014

PubMed Central

Zhuge, Jian; Vail, Eric; Bush, Jeffrey L.; Singelakis, Lauren; Huang, Weihua; Nolan, Sheila M.; Haas, Janet P.; Engel, Helen; Della Posta, Millicent; Yoon, Esther C.; Fallon, John T.

2015-01-01

An outbreak of severe respiratory illness associated with enterovirus D68 (EV-D68) infection was reported in mid-August 2014 in the United States. In this study, we evaluated the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (rRT-PCR) that was recently developed by the Centers for Disease Control and Prevention in clinical samples. Nasopharyngeal (NP) swab specimens from patients in a recent outbreak of respiratory illness in the lower Hudson Valley, New York State, were collected and examined for the presence of human rhinovirus or enterovirus using the FilmArray Respiratory Panel (RP) assay. Samples positive by RP were assessed using EV-D68 rRT-PCR, and the data were compared to results from sequencing analysis of partial VP1 and 5′ untranslated region (5′-UTR) sequences of the EV genome. A total of 285 RP-positive NP specimens (260 from the 2014 outbreak and 25 from 2013) were analyzed by rRT-PCR; EV-D68 was detected in 74 of 285 (26.0%) specimens examined. Data for comparisons between rRT-PCR and sequencing analysis were obtained from 194 NP specimens. EV-D68 detection was confirmed by sequencing analysis in 71 of 74 positive and in 1 of 120 randomly selected negative specimens by rRT-PCR. The EV-D68 rRT-PCR showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. Our data suggest that the EV-D68 rRT-PCR is a reliable assay for detection of EV-D68 in clinical samples and has a potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories. PMID:25854481

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

PubMed Central

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

PubMed

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR.

PubMed

Ma, Yue-Jiao; Sun, Xiao-Hong; Xu, Xiao-Yan; Zhao, Yong; Pan, Ying-Jie; Hwang, Cheng-An; Wu, Vivian C H

2015-01-01

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh+, ATCC33846-tdh+, ATCC33847-tdh+, ATCC17802-trh+, and F13-environmental strain-tdh+) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.

A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens.

PubMed

Lung, Jrhau; Lin, Yu-Ching; Hung, Ming-Szu; Jiang, Yuan Yuan; Lee, Kuan-Der; Lin, Paul Yann; Tsai, Ying Huang

2016-04-01

ALK fusion gene is an oncogenic driver in lung cancer with low prevalence, which can be ameliorated by crizotinib. Currently, ALK fusion gene can be diagnosed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), but inconstistnt results between the two methods are encountered regularly. To make the ALK fusion gene screening more efficient and to provide a simple solution to clarify the discrepancy between FISH and IHC results, a sensitive TaqMan-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was established. The 3-plex TaqMan-based RT-qPCR assay was established and performed on 102 archived formalin-fixed, paraffin-embedded (FFPE) NSCLC samples to detect ALK rearrangement and overexpression. Break-apart FISH and automatic immunohistochemistry based ALK assays were performed side by side using tissue microarray. The RT-qPCR was performed successfully for 80 samples and 10 of them showed positive signals. Three out of the 10 qPCR positive cases were further confirmed by FISH and IHC test. Two others were IHC positive and FISH negative, and expressed full-length ALK transcript. The rest were neither FISH nor IHC positive and their ALK expression level was significantly lower than those FISH or IHC positive cases. Our RT-qPCR assay demonstrates that the capability and reliability of ALK detection is comparable to FISH and IHC, but it is more effective at discriminating ALK rearrangement from overexpression. The RT-qPCR assay easily clarifies the discrepancy between FISH and IHC, and can be incorporated into routine ALK screening for lung cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Analysis of differential gene expression by bead-based fiber-optic array in nonfunctioning pituitary adenomas.

PubMed

Jiang, Z; Gui, S; Zhang, Y

2011-05-01

Nonfunctioning pituitary adenomas (NFPAs) are relatively common, accounting for 30% of all pituitary adenomas; however, their pathogenesis remains enigmatic. To explore the possible pathogenesis of NFPAs, we used fiber-optic BeadArray to examine gene expression in 5 NFPAs compared with 3 normal pituitaries. 4 differentially expressed genes were chosen randomly for validation by reverse transcriptase-real time quantitative polymerase chain reaction (RT-qPCR). We then analyzed the differentially expressed gene profile with Kyoto Encyclopedia of Genes and Genomes (KEGG). The array analysis indentified significant increases in the expression of 1,402 genes and 383 expressed sequence tags (ESTs), and decreases in 1,697 genes and 113 ESTs in the NFPAs. Bioinformatic and pathway analysis showed that the genes HIGD1B, FAM5C, PMAIP1 and the pathway cell-cycle regulation may play an important role in tumorigenesis and progression of NFPAs. Our data suggest fiber-optic BeadArray combined with pathway analysis of differential gene expression profile appears to be a valid approach for investigating the pathogenesis of tumors. © Georg Thieme Verlag KG Stuttgart · New York.

Comparative Diagnosis of Human Bocavirus 1 Respiratory Infection With Messenger RNA Reverse-Transcription Polymerase Chain Reaction (PCR), DNA Quantitative PCR, and Serology.

PubMed

Xu, Man; Arku, Benedict; Jartti, Tuomas; Koskinen, Janne; Peltola, Ville; Hedman, Klaus; Söderlund-Venermo, Maria

2017-05-15

Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection. Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection. By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%). A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma.

PubMed

Hamatake, Makiko; Nishizawa, Masako; Yamamoto, Naoki; Kato, Shingo; Sugiura, Wataru

2007-06-01

An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.

Sensitivity of Small RNA-Based Detection of Plant Viruses.

PubMed

Santala, Johanna; Valkonen, Jari P T

2018-01-01

Plants recognize unrelated viruses by the antiviral defense system called RNA interference (RNAi). RNAi processes double-stranded viral RNA into small RNAs (sRNAs) of 21-24 nucleotides, the reassembly of which into longer strands in silico allows virus identification by comparison with the sequences available in databases. The aim of this study was to compare the virus detection sensitivity of sRNA-based virus diagnosis with the established virus species-specific polymerase chain reaction (PCR) approach. Viruses propagated in tobacco plants included three engineered, infectious clones of Potato virus A (PVA), each carrying a different marker gene, and an infectious clone of Potato virus Y (PVY). Total RNA (containing sRNA) was isolated and subjected to reverse-transcription real-time PCR (RT-RT-PCR) and sRNA deep-sequencing at different concentrations. RNA extracted from various crop plants was included in the reactions to normalize RNA concentrations. Targeted detection of selected viruses showed a similar threshold for the sRNA and reverse-transcription quantitative PCR (RT-qPCR) analyses. The detection limit for PVY and PVA by RT-qPCR in this study was 3 and 1.5 fg of viral RNA, respectively, in 50 ng of total RNA per PCR reaction. When knowledge was available about the viruses likely present in the samples, sRNA-based virus detection was 10 times more sensitive than RT-RT-PCR. The advantage of sRNA analysis is the detection of all tested viruses without the need for virus-specific primers or probes.

Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

PubMed

Suryavanshi, Moushumi; Mehta, Anurag; Jaipuria, Jiten; Kumar, Dushyant; Vishwakarma, Gayatri; Panigrahi, Manoj Kumar; Verma, Haristuti; Saifi, Mumtaz; Sharma, Sanjeev; Tandon, Simran; Doval, D C; Das, Bhudev C

2018-02-09

IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients would have benefited from RT-PCR testing if it was used as a first-line test. If RT-PCR would have been used as a second-line test among those with IHC score 2 (n = 43), then only 6 patients would have been assigned a conclusive RT-PCR category (category 1 or 3) translating to a clinical benefit of 14% (6/43) as a second-line test. As a second-line test it had 51% probability to prove more cost-effective than the conventional pathway, provided the cost of RT-PCR was 0.4 times the cost of IHC. Also in a three-step pathway, RT-PCR upfront would have 56% probability of higher cost-benefit provided the cost of RT-PCR was 0.1 times the cost of IHC. RT-PCR results were found to be suboptimal to IHC in terms of discriminative ability and clinical benefit; thus, it is unlikely to replace IHC as a first-line test in the near future.

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors

PubMed Central

Thakore, Pratiksha I.; Gersbach, Charles A.

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy. PMID:26443215

Retention of Rotavirus Infectivity in Mussels Heated by Using the French Recipe Moules Marinières.

PubMed

Souza, Doris Sobral Marques; Miura, Takayuki; Le Mennec, Cécile; Barardi, Célia Regina Monte; Le Guyader, Françoise S

2015-11-01

To evaluate the persistence of infectious virus after heating, mussels contaminated with a rotavirus strain were prepared following the French recipe moules marinières (mariner's mussels). Rotavirus was then quantified by real-time quantitative PCR (RT-qPCR) and a cell culture infectivity assay. Results showed the persistence of infectious virus after 3 min of cooking. After 5 min, when no infectious virus could be detected, the RT-qPCR approach showed a 1-log decrease compared with concentrations detected after 1 min of cooking.

Clinical Impact of Hepatitis C Infection in Military Active Duty Women

DTIC Science & Technology

1996-07-01

presence of viral RNA (HCV RNA) by qualitative and quantitative assays: RT PCR (primers 5’ UTR) bDNA (Chiron Laboratories, Emeryville, CA) Amplicor...PCR) bDNA Eq/mL x 10’ Amplicor Copies/mL 1 Positive 27.11 (Positive) 340,135 (Positive) 2 Negative < 3.5 (Negative) (Negative) 3 Negative < 3.5...the advantage of measuring titer of RNA in serum, however the sensitivity is low. 8 TABLE 3 Comparison of the Sensitivity of HCV RNA by RT-PCR, bDNA

Large-scale screening and molecular characterization of EML4-ALK fusion variants in archival non-small-cell lung cancer tumor specimens using quantitative reverse transcription polymerase chain reaction assays.

PubMed

Li, Tianhong; Maus, Martin K H; Desai, Sonal J; Beckett, Laurel A; Stephens, Craig; Huang, Eric; Hsiang, Jack; Zeger, Gary; Danenberg, Kathleen D; Astrow, Stephanie H; Gandara, David R

2014-01-01

The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non-small-cell lung cancer (NSCLC) patients. We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23-89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

PubMed Central

Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A

2008-01-01

Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability. PMID:19102748

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4+ Level

PubMed Central

de Freitas, Vera Lúcia Teixeira; da Silva, Sheila Cristina Vicente; Sartori, Ana Marli; Bezerra, Rita Cristina; Westphalen, Elizabeth Visone Nunes; Molina, Tatiane Decaris; Teixeira, Antonio R. L.; Ibrahim, Karim Yaqub; Shikanai-Yasuda, Maria Aparecida

2011-01-01

Background Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4+ cells or the CD4+/CD8+ ratio. Conclusions qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4+ cells/mm3 and the CD4+/CD8+ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy. PMID:21912712

Altered retinal microRNA expression profiles in early diabetic retinopathy: an in silico analysis.

PubMed

Xiong, Fen; Du, Xinhua; Hu, Jianyan; Li, Tingting; Du, Shanshan; Wu, Qiang

2014-07-01

MicroRNAs (miRNAs) - as negative regulators of target genes - are associated with various human diseases, but their precise role(s) in diabetic retinopathy (DR) remains to be elucidated. The aim of this study was to elucidate the involvement of miRNAs in early DR using in silico analysis to explore their gene expression patterns. We used the streptozotocin (STZ)-induced diabetic rat to investigate the roles of miRNAs in early DR. Retinal miRNA expression profiles from diabetic versus healthy control rats were examined by miRNA array analysis. Based on several bioinformatic systems, specifically, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified signatures of the potential pathological processes, gene functions, and signaling pathways that are influenced by dysregulated miRNAs. We used quantitative real-time polymerase chain reaction (qRT-PCR) to validate six (i.e. those with significant changes in expression levels) of the 17 miRNAs that were detected in the miRNA array. We also describe the significant role of the miRNA-gene network, which is based on the interactions between miRNAs and target genes. GO analysis of the 17 miRNAs detected in the miRNA array analysis revealed the most prevalent miRNAs to be those related to biological processes, olfactory bulb development and axonogenesis. These miRNAs also exert significant influence on additional pathways, including the mitogen-activated protein and calcium signaling pathways. Six of the seventeen miRNAs were chosen for qRT-PCR validation. With the exception of a slight difference in miRNA-350, our results are in close agreement with the differential expressions detected by array analysis. This study, which describes miRNA expression during the early developmental phases of DR, revealed extensive miRNA interactions. Based on both their target genes and signaling pathways, we suggest that miRNAs perform critical regulatory functions during the early stages of DR evolution.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus.

PubMed

Zhang, Qingli; Standish, Isaac; Winters, Andrew D; Puzach, Corey; Ulferts, Rachel; Ziebuhr, John; Faisal, Mohamed

2014-06-01

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV. Copyright © 2014 Elsevier B.V. All rights reserved.

Premalignant Genetic and Epigenetic Alterations in Tubal Epithelium from Women with BRCA1 Mutations

DTIC Science & Technology

2010-10-14

Homo sapiens , clone IMAGE:5590287, mRNA Unknown 1.8 .0044 3.0...RT-PCR W T -F T B 1- F T B 1 C an ce r 0 2 4 6 8 10 R el at iv e Q u an ti fi ca ti o n CDKN1C Array W T -F T B 1- F T B 1 C an ce r 0 100 200 300...S p o t In te n si ty CDKN1C RT-PCR W T -F T B 1- F T B 1 C an ce r 0.0 0.2 0.4 0.6 0.8 R el at iv e Q u an ti fi ca ti o n CYP3A5 Array W T

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

PubMed

Pinches, Mark D G; Helps, Christopher R; Gruffydd-Jones, Tim J; Egan, Kathy; Jarrett, Oswald; Tasker, Séverine

2007-02-01

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

Detection and quantification of pestivirus in experimentally infected pregnant ewes and their progeny.

PubMed

Hurtado, Ana; Sanchez, Isbene; Bastida, Felix; Minguijón, Esmeralda; Juste, Ramón A; García-Pérez, Ana L

2009-11-05

Border disease virus (BDV) causes important reproductive losses, and eradication strategies focus on the identification and removal of persistently infected animals arising after in uterine infection. BDV infection dynamics were studied in 13 ewes experimentally infected with BDV-4 genotype at 3 phases of pregnancy [days 108 (group A), 76 (group B) and 55 (group C)] by quantification of viral RNA in blood collected on days -1 to parturition using quantitative real-time RT-PCR (qRT-PCR). Viral RNA loads were also measured in blood/foetal fluid and tissue samples from their offspring at lambing (3 foetuses, 7 stillborns, 15 lambs). qRT-PCR results were compared with those obtained by conventional RT-PCR and used to predict persistent infections. Viral RNA was detected in the ewes between days 2-15 p.i. The viraemia reached its highest peak between days 6-7 p.i. with a second peak at days 11-12 p.i. qRT-PCR was significantly faster to perform (less than 1 h) than conventional RT-PCR and detected BDV RNA in more ewes, being detection more continuous and prolonged in time. The virus was detected in peripheral blood in a higher percentage of lambs than in tissues, where differences in viral genome copies were more marked. Skin and cerebral cortex showed the highest viral RNA loads, and spleen and spinal cord the lowest. High viral RNA loads were observed in several animals in group B and all in group C, infected during middle and early foetal development, respectively, but also in one lamb from group A, infected during late foetal development. Serology and viral genome copy number estimates in blood and tissues were used to establish a quantitative cut-off threshold for transient viraemia. Viral RNA quantification showed potential for the discrimination between persistent infections and transient viraemia using single-time point blood sampling and raised questions regarding foetal immune system development and the occurrence of persistent infections.

Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR.

PubMed

Gan, W; Zhou, X; Yang, H; Chen, H; Qiao, J; Khan, S H; Yang, L; Yin, X; Zhao, D

2013-08-03

The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.

Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia.

PubMed

Wattjes, M P; Krauter, J; Nagel, S; Heidenreich, O; Ganser, A; Heil, G

2000-02-01

The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages.

The Role of eIF4E Activity in Breast Cancer

DTIC Science & Technology

2010-08-01

ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less product...have previously shown that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem

Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

PubMed

Roh, Ha-Jung; Hilt, Deborah A; Jackwood, Mark W

2014-09-01

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs.

Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

PubMed

Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

2015-01-01

Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by Ozone Disinfection of Water

PubMed Central

Shin, Gwy-Am; Sobsey, Mark D.

2003-01-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log10 within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone. PMID:12839770

Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water.

PubMed

Shin, Gwy-Am; Sobsey, Mark D

2003-07-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5 degrees C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log(10) within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.

Modeling qRT-PCR dynamics with application to cancer biomarker quantification.

PubMed

Chervoneva, Inna; Freydin, Boris; Hyslop, Terry; Waldman, Scott A

2017-01-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.

Methods for Characterization of Alternative RNA Splicing.

PubMed

Harvey, Samuel E; Cheng, Chonghui

2016-01-01

Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

Single-pair fluorescence resonance energy transfer analysis of mRNA transcripts for highly sensitive gene expression profiling in near real time.

PubMed

Peng, Zhiyong; Young, Brandon; Baird, Alison E; Soper, Steven A

2013-08-20

Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with online single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10-10,000 copies) with an R(2) = 0.9995 for RT-LDR/spFRET and R(2) = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a two thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time-sensitive therapeutics.

Single-Pair Fret Analysis of mRNA Transcripts for Highly Sensitive Gene Expression Profiling in Near Real Time

PubMed Central

Peng, Zhiyong; Young, Brandon; Baird, Alison E.; Soper, Steven A.

2013-01-01

Expression analysis of mRNAs transcribed from certain genes can be used as important sources of biomarkers for in vitro diagnostics. While the use of reverse transcription quantitative PCR (RT-qPCR) can provide excellent analytical sensitivity for monitoring transcript numbers, more sensitive approaches for expression analysis that can report results in near real-time are needed for many critical applications. We report a novel assay that can provide exquisite limits-of-quantitation and consists of reverse transcription (RT) followed by a ligase detection reaction (LDR) with single-pair fluorescence resonance energy transfer (spFRET) to provide digital readout through molecular counting. For this assay, no PCR was employed, which enabled short assay turnaround times. To facilitate implementation of the assay, a cyclic olefin copolymer (COC) microchip, which was fabricated using hot embossing, was employed to carry out the LDR in a continuous flow format with on-line single-molecule detection following the LDR. As demonstrators of the assay's utility, MMP-7 mRNA was expression profiled from several colorectal cancer cell lines. It was found that the RT-LDR/spFRET assay produced highly linear calibration plots even in the low copy number regime. Comparison to RT-qPCR indicated a better linearity over the low copy number range investigated (10 − 10,000 copies) with an R2 = 0.9995 for RT-LDR/spFRET and R2 = 0.98 for RT-qPCR. In addition, differentiating between copy numbers of 10 and 50 could be performed with higher confidence using RT-LDR/spFRET. To demonstrate the short assay turnaround times obtainable using the RT-LDR/spFRET assay, a 2 thermal cycle LDR was carried out on amphiphysin gene transcripts that can serve as important diagnostic markers for ischemic stroke. The ability to supply diagnostic information on possible stroke events in short turnaround times using RT-LDR/spFRET will enable clinicians to treat patients effectively with appropriate time-sensitive therapeutics. PMID:23869556

Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries.

PubMed

Choi, Eun-Jung; Lee, Sung-Hoon; Kim, Young-Jae

2009-03-01

Streptococcus mutans and Streptococcus sobrinus are closely associated with the development of early childhood caries (ECC). Recently, quantitative real-time polymerase chain reaction (qRT-PCR) has been used for rapid and accurate quantification of these bacterial species. This study aims to detect quantitatively the levels of S. mutans and S. sobrinus in plaque samples by qRT-PCR, and to assess their association with the prevalence of ECC in Korean preschool children. One hundred and five children (71 months old or younger) were examined and classified into three groups (caries-free, ECC, severe ECC). Dental plaque samples were collected and qRT-PCR was conducted using oligonucleotide primers specific for glucosyltransferase gene (S. mutans-gtfB, S. sobrinus-gtfU) and universal primer. Pearson's correlation test was conducted to evaluate the relationship between the dmfs (decayed, missing, or filled surfaces primary teeth) scores and the microbiological findings. There was a significant difference between the levels of S. mutans and S. sobrinus in the plaque samples of the three groups (P < 0.05). The proportion of S. sobrinus to S. mutans showed strong correlation to the dmfs scores (r = 0.748, P < 0.05). The qRT-PCR results of this study showed that children with ECC had higher level of S. mutans and S. sobrinus in their dental plaque samples. The children with higher ratio of S. sobrinus to S. mutans in their dental plaque showed higher incidence of ECC.

Zika Virus Infection of the Human Glomerular Cells: Implications for Viral Reservoirs and Renal Pathogenesis.

PubMed

Alcendor, Donald J

2017-07-15

Zika virus (ZIKV) infection in the human renal compartment has not been reported. Several clinical reports have describe high-level persistent viral shedding in the urine of infected patients, but the associated mechanisms have not been explored until now. The current study examined cellular components of the glomerulus of the human kidney for ZIKV infectivity. I infected primary human podocytes, renal glomerular endothelial cells (GECs), and mesangial cells with ZIKV. Viral infectivity was analyzed by means of microscopy, immunofluorescence, real-time reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qRT-PCR), and the proinflammatory cytokines interleukin 1β, interferon β, and RANTES (regulated on activation of normal T cells expressed and secreted) were assessed using qRT-PCR. I show that glomerular podocytes, renal GECs, and mesangial cells are permissive for ZIKV infection. ZIKV infectivity was confirmed in all 3 cell types by means of immunofluorescence staining, RT-PCR, and qRT-PCR, and qRT-PCR analysis revealed increased transcriptional induction of interleukin 1β, interferon β, and RANTES in ZIKV-infected podocytes at 72 hours, compared with renal GECs and mesangial cells. The findings of this study support the notion that the glomerulus may serve as an amplification reservoir for ZIKV in the renal compartment. The impact of ZIKV infection in the human renal compartment is unknown and will require further study. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Quantitative Real-time Polymerase Chain Reaction for Enteropathogenic Escherichia coli: A Tool for Investigation of Asymptomatic Versus Symptomatic Infections

PubMed Central

Barletta, Francesca; Mercado, Erik; Ruiz, Joaquim; Ecker, Lucie; Lopez, Giovanni; Mispireta, Monica; Gil, Ana I.; Lanata, Claudio F.; Cleary, Thomas G.

2011-01-01

Background. Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon. Methods. EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined. Results. The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77–1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10–87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006). Conclusions. EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity. PMID:22028433

Quantitative real time RT-PCR study of pathogen-induced gene expression in rock bream (Oplegnathus fasciatus): internal controls for data normalization.

PubMed

Zhang, Bao-cun; Sun, Li; Xiao, Zhi-zhong; Hu, Yong-hua

2014-06-01

Rock bream Oplegnathus fasciatus is an important economic fish species. In this study, we evaluated the appropriateness of six housekeeping genes as internal controls for quantitative real-time PCR (RT-qPCR) analysis of gene expression in rock bream before and after pathogen infection. The expression of the selected genes in eight tissues infected with Vibrio alginolyticus or megalocytivirus was determined by RT-qPCR, and the PCR data were analyzed with geNorm and NormFinder algorithms. The results showed that before pathogen infection, mediator of RNA polymerase II transcription subunit 8 and β-actin were ranked as the most stable genes across the examined tissues. After bacterial or viral infection, the stabilities of the housekeeping genes varied to significant extents in tissue-dependent manners, and no single pair of genes was identified as suitable references for all tissues for either of the pathogen stimuli. In addition, for the majority of tissues, the most stable genes during bacterial infection differed from those during viral infection. Nevertheless, optimum reference genes were identified for each tissue under different conditions. Taken together, these results indicate that tissue type and the nature of the infectious agent used in the study can all influence the choice of normalization factors, and that the optimum reference genes identified in this study will provide a useful guidance for the selection of internal controls in future RT-PCR study of gene expression in rock bream. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation

PubMed Central

Schliep, M.; Pernice, M.; Sinutok, S.; Bryant, C. V.; York, P. H.; Rasheed, M. A.; Ralph, P. J.

2015-01-01

Seagrass meadows are threatened by coastal development and global change. In the face of these pressures, molecular techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) have great potential to improve management of these ecosystems by allowing early detection of chronic stress. In RT-qPCR, the expression levels of target genes are estimated on the basis of reference genes, in order to control for RNA variations. Although determination of suitable reference genes is critical for RT-qPCR studies, reports on the evaluation of reference genes are still absent for the major Australian species Zostera muelleri subsp. capricorni (Z. muelleri). Here, we used three different software (geNorm, NormFinder and Bestkeeper) to evaluate ten widely used reference genes according to their expression stability in Z. muelleri exposed to light limitation. We then combined results from different software and used a consensus rank of four best reference genes to validate regulation in Photosystem I reaction center subunit IV B and Heat Stress Transcription factor A- gene expression in Z. muelleri under light limitation. This study provides the first comprehensive list of reference genes in Z. muelleri and demonstrates RT-qPCR as an effective tool to identify early responses to light limitation in seagrass. PMID:26592440

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

PubMed

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Multiplex titration RT-PCR: rapid determination of gene expression patterns for a large number of genes

NASA Technical Reports Server (NTRS)

Nebenfuhr, A.; Lomax, T. L.

1998-01-01

We have developed an improved method for determination of gene expression levels with RT-PCR. The procedure is rapid and does not require extensive optimization or densitometric analysis. Since the detection of individual transcripts is PCR-based, small amounts of tissue samples are sufficient for the analysis of expression patterns in large gene families. Using this method, we were able to rapidly screen nine members of the Aux/IAA family of auxin-responsive genes and identify those genes which vary in message abundance in a tissue- and light-specific manner. While not offering the accuracy of conventional semi-quantitative or competitive RT-PCR, our method allows quick screening of large numbers of genes in a wide range of RNA samples with just a thermal cycler and standard gel analysis equipment.

Design of primers and probes for quantitative real-time PCR methods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Córdoba, Juan J; Andrade, María J

2015-01-01

Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

PubMed Central

Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

2016-01-01

Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes. PMID:27242878

A tool for design of primers for microRNA-specific quantitative RT-qPCR.

PubMed

Busk, Peter K

2014-01-28

MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets. I have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies. The software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories.

PubMed

Domingo, Cristina; Patel, Pranav; Yillah, Jasmin; Weidmann, Manfred; Méndez, Jairo A; Nakouné, Emmanuel Rivalyn; Niedrig, Matthias

2012-12-01

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR

PubMed Central

2014-01-01

Background Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly. Results We took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf). Conclusions We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB. PMID:24533511

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

PubMed

Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Predictive value of PD-L1 based on mRNA level in the treatment of stage IV melanoma with ipilimumab.

PubMed

Brüggemann, C; Kirchberger, M C; Goldinger, S M; Weide, B; Konrad, A; Erdmann, M; Schadendorf, D; Croner, R S; Krähenbühl, L; Kähler, K C; Hafner, C; Leisgang, W; Kiesewetter, F; Dummer, R; Schuler, G; Stürzl, M; Heinzerling, L

2017-10-01

PD-L1 is established as a predictive marker for therapy of non-small cell lung cancer with pembrolizumab. Furthermore, PD-L1 positive melanoma has shown more favorable outcomes when treated with anti-PD1 antibodies and dacarbazine compared to PD-L1 negative melanoma. However, the role of PD-L1 expression with regard to response to checkpoint inhibition with anti-CTLA-4 is not clear, yet. In addition, the lack of standardization in the immunohistochemical assessment of PD-L1 makes the comparison of results difficult. In this study, we investigated the PD-L1 gene expression with a new fully automated technique via RT-PCR and correlated the findings with the response to the anti-CTLA-4 antibody ipilimumab. Within a retrospective multi-center trial, PD-L1 gene expression was evaluated in 78 melanoma patients in a total of 111 pre-treatment tumor samples from 6 skin cancer centers and analyzed with regard to response to ipilimumab. For meaningful statistical analysis, the cohort was enriched for responders with 30 responders and 48 non-responders. Gene expression was assessed by quantitative RT-PCR after extracting mRNA from formalin-fixed paraffin embedded tumor tissue and correlated with results from immunohistochemical (IHC) stainings. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab.

[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].

PubMed

Hang, Xiao-Tong; Li, Jian-Dong; Zhang, Quan-Fu; Li, Chuan; Zhang, Shuo; Liang, Mi-Fang; Li, De-Xin

2010-02-01

Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.

miRNA expression profiling in formalin-fixed paraffin-embedded endometriosis and ovarian cancer samples

PubMed Central

Braicu, Ovidiu-Leonard; Budisan, Liviuta; Buiga, Rares; Jurj, Ancuta; Achimas-Cadariu, Patriciu; Pop, Laura Ancuta; Braicu, Cornelia; Irimie, Alexandru; Berindan-Neagoe, Ioana

2017-01-01

Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19–22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition. PMID:28894379

Measuring and mitigating inhibition during real-time, quantitative PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

USDA-ARS?s Scientific Manuscript database

Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, ...

Recommended reference genes for quantitative PCR analysis in soybean have variable stabilities during diverse biotic stresses

USDA-ARS?s Scientific Manuscript database

For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is n...

Expression of stress/defense-related genes in barley grown under space environment

NASA Astrophysics Data System (ADS)

Sugimoto, Manabu; Shagimardanova, Elena; Gusev, Oleg; Bingham, Gail; Levinskikh, Margarita; Sychev, Vladimir

Plants are exposed to the extreme environment in space, especially space radiation is suspected to induce oxidative stress by generating high-energy free radicals and microgravity would enhance the effect of space radiation, however, current understandings of plant growth and responses on this synergistic effect of radiation and microgravity is limited to a few experiments. In this study, expression of stress/defense-related genes in barley grown under space environment was analyzed by RT-PCR and DNA microarray experiments to understand plant responses and adaptation to space environment and to develop the space stress-tolerant plants. The seeds of barley, Hordeum vulgare L. cv. Haruna nijo, kept in the international space station (ISS) over 4 months, were germinated after 3 days of irrigation in LADA plant growth chamber onboard Russian segment of ISS and the final germination ratio was over 90 %. The height of plants was about 50 to 60 cm and flag leaf has been opened after 26 days of irrigation under 24 hr lighting, showing the similar growth to ground-grown barley. Expression levels of stress/defense-related genes in space-grown barley were compared to those in ground-grown barley by semi-quantitative RT-PCR. In 17 stress/defense-related genes that are up-regulated by oxidative stress or other abiotic stress, only catalase, pathogenesis-related protein 13, chalcone synthase, and phenylalanine ammonia-lyase genes were increased in space-grown barley. DNA microarrya analysis with the GeneChip Barley Genome Array showed the similar expression profiles of the stress/defense-related genes to those by RT-PCR experiment, suggesting that the barley germinated and grown in LADA onboard ISS is not damaged by space environment, especially oxidative stress induced by space radiation and microgravity.

Novel in Vitro Modification of Bone for an Allograft with Improved Toughness Osteoconductivity

DTIC Science & Technology

2015-06-01

osteocalcin, Runx2, and col1a1 by RT-PCR. Spectrophotometry and fluorescence microscopy were used to quantify AGEs. 2. KEYWORDS Fracture toughness, R...markers (alkaline phosphatase, osteocalcin, RUNX2 and COL1A1 ) Completed Task 10 Data analysis, publications, reports Completed Task 1. Retrieval...FEMALE 25 Task 9. Measure expression of molecular markers of mineralization, osteocalcin, RUNX2 and COL1A1 using quantitative RT-PCR with specific

Novel in Vitro Modification of Bone for an Allograft with Improved Toughness Osteoconductivity

DTIC Science & Technology

2014-04-01

of bone-characteristic genes, osteocalcin, Runx2, and col1a1 by RT-PCR. High-performance liquid chromatography and fluorescence microscopy will be...of molecular markers of mineralization, osteocalcin, Runx2 and col1a1 using quantitative RT-PCR with specific primers. (Months 8-15.) The purpose...bone specific Collagen, type I, alpha 1 ( COL1A1 ) Associated with cell adhesion, proliferation and differentiation of the osteoblast phenotype and

Novel in Vitro Modification of Bone for an Allograft with Improved Toughness Osteoconductivity

DTIC Science & Technology

2013-10-01

col1a1 by RT-PCR. High-performance liquid chromatography and fluorescence microscopy will be used to quantify AGEs and crosslinks. BODY The...molecular markers of mineralization, osteocalcin, Runx2 and col1a1 using quantitative RT-PCR with specific primers. (Months 14-15.) 5a. Preperation of...cellular activity and differentiation but not bone specific Collagen, type I, alpha 1 ( COL1A1 ) Associated with cell adhesion, proliferation and

The Role of elF4E Activity in Breast Cancer

DTIC Science & Technology

2011-08-01

protein; ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...Reactions were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less...that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem-loop structure6. This

Selection of internal reference genes for normalization of quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in the canine brain and other organs.

PubMed

Park, Sang-Je; Huh, Jae-Won; Kim, Young-Hyun; Lee, Sang-Rae; Kim, Sang-Hyun; Kim, Sun-Uk; Kim, Heui-Soo; Kim, Min Kyu; Chang, Kyu-Tae

2013-05-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive technique for quantifying gene expression. To analyze qRT-PCR data accurately, suitable reference genes that show consistent expression patterns across different tissues and experimental conditions should be selected. The objective of this study was to obtain the most stable reference genes in dogs, using samples from 13 different brain tissues and 10 other organs. 16 well-known candidate reference genes were analyzed by the geNorm, NormFinder, and BestKeeper programs. Brain tissues were derived from several different anatomical regions, including the forebrain, cerebrum, diencephalon, hindbrain, and metencephalon, and grouped accordingly. Combination of the three different analyses clearly indicated that the ideal reference genes are ribosomal protien S5 (RPS5) in whole brain, RPL8 and RPS5 in whole body tissues, RPS5 and RPS19 in the forebrain and cerebrum, RPL32 and RPS19 in the diencephalon, GAPDH and RPS19 in the hindbrain, and MRPS7 and RPL13A in the metencephalon. These genes were identified as ideal for the normalization of qRT-PCR results in the respective tissues. These findings indicate more suitable and stable reference genes for future studies of canine gene expression.

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Identification of downy mildew resistance gene candidates by positional cloning in maize (Zea mays subsp. mays; Poaceae)1

PubMed Central

Kim, Jae Yoon; Moon, Jun-Cheol; Kim, Hyo Chul; Shin, Seungho; Song, Kitae; Kim, Kyung-Hee; Lee, Byung-Moo

2017-01-01

Premise of the study: Positional cloning in combination with phenotyping is a general approach to identify disease-resistance gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combined strategy to improve the identification of disease-resistance gene candidates. Methods and Results: Downy mildew (DM)–resistant maize was selected from five cultivars using a spreader row technique. Positional cloning and bioinformatics tools were used to identify the DM-resistance quantitative trait locus marker (bnlg1702) and 47 protein-coding gene annotations. Eventually, five DM-resistance gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative reverse-transcription PCR (RT-PCR) without fine mapping of the bnlg1702 locus. Conclusions: The combined protocol with the spreader row technique, quantitative trait locus positional cloning, and quantitative RT-PCR was effective for identifying DM-resistance candidate genes. This cloning approach may be applied to other whole-genome-sequenced crops or resistance to other diseases. PMID:28224059

Toxoplasma gondii infections in chickens - performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods.

PubMed

Schares, G; Koethe, M; Bangoura, B; Geuthner, A-C; Randau, F; Ludewig, M; Maksimov, P; Sens, M; Bärwald, A; Conraths, F J; Villena, I; Aubert, D; Opsteegh, M; Van der Giessen, J

2018-05-19

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100 times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes

USGS Publications Warehouse

Purcell, M.K.; Laing, K.J.; Woodson, J.C.; Thorgaard, G.H.; Hansen, J.D.

2009-01-01

The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-??) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-?? gene (rtIFN-??2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in na??ve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-??1 and rtIFN-??2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-??2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection. ?? 2008 Elsevier Ltd.

Reliable Gene Expression Measurements from Fine Needle Aspirates of Pancreatic Tumors

PubMed Central

Anderson, Michelle A.; Brenner, Dean E.; Scheiman, James M.; Simeone, Diane M.; Singh, Nalina; Sikora, Matthew J.; Zhao, Lili; Mertens, Amy N.; Rae, James M.

2010-01-01

Background and aims: Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression. Methods: Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft. Results: RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance. Conclusions: The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens “informative” for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases. PMID:20709792

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

PubMed Central

2014-01-01

Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1α have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided. PMID:25048176

Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

PubMed

Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William

2011-12-01

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. Published by Elsevier B.V.

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.

PubMed

Jothikumar, Narayanan; Cromeans, Theresa L; Robertson, Betty H; Meng, X J; Hill, Vincent R

2006-01-01

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes

PubMed Central

Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes. PMID:29293626

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes.

PubMed

Xiang, Quanju; Li, Jin; Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.

Early Detection of Dengue Virus by Use of Reverse Transcription-Recombinase Polymerase Amplification

PubMed Central

Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Danlami, Mohammed Bashar; Shu, Meng-Hooi; Johari, Jefree; Hooi, Poh-Sim; Brooks, David; Piepenburg, Olaf; Nentwich, Oliver; Wilder-Smith, Annelies; Franco, Leticia; Tenorio, Antonio

2015-01-01

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue. PMID:25568438

Quantitative analysis of periodontal pathogens by ELISA and real-time polymerase chain reaction.

PubMed

Hamlet, Stephen M

2010-01-01

The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.

Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

PubMed

Memon, Atta Muhammad; Bhuyan, Anjuman Ara; Chen, Fangzhou; Guo, Xiaozhen; Menghwar, Harish; Zhu, Yinxing; Ku, Xugang; Chen, Shuhua; Li, Zhonghua; He, Qigai

2017-05-01

Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 10 2.5 TCID 50 /mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

PubMed

Osman, Fatima; Dang, Tyler; Bodaghi, Sohrab; Vidalakis, Georgios

2017-07-01

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs. Copyright © 2017. Published by Elsevier B.V.

Molecular epidemiology of human metapneumovirus in Ireland.

PubMed

Carr, Michael J; Waters, Allison; Fenwick, Fiona; Toms, Geoffrey L; Hall, William W; O'Kelly, Edwin

2008-03-01

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real-time RT-PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real-time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub-clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT-PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44-8.15). Statistical analysis of quantitative RT-PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates (P < 0.03).

Comparative evaluation of serum, FTA filter-dried blood and oral fluid as sample material for PRRSV diagnostics by RT-qPCR in a small-scale experimental study.

PubMed

Steinrigl, Adolf; Revilla-Fernández, Sandra; Wodak, Eveline; Schmoll, Friedrich; Sattler, Tatjana

2014-01-01

Recently, research into alternative sample materials, such as oral fluid or filter-dried blood has been intensified, in order to facilitate cost-effective and animal-friendly sampling of individuals or groups of pigs for diagnostic purposes. The objective of this study was to compare the sensitivity of porcine reproductive and respiratory syndrome virus (PRRSV)-RNA detection by reverse transcription quantitative real-time PCR (RT-qPCR) in serum, FTA filter-dried blood and oral fluid sampled from individual pigs. Ten PRRSV negative pigs were injected with an EU-type PRRSV live vaccine. Blood and oral fluid samples were taken from each pig before, and 4, 7, 14 and 21 days after vaccination. All samples were then analyzed by PRRSV RT-qPCR. In serum, eight often pigs tested RT-qPCR positive at different time points post infection. Absolute quantification showed low serum PRRSV-RNA loads in most samples. In comparison to serum, sensitivity of PRRSV-RNA detection was strongly reduced in matched FTA filter-dried blood and in oral fluid from the same pigs. These results indicate that with low PRRSV-RNA loads the diagnostic sensitivity of PRRSV-RNA detection by RT-qPCR achieved with serum is currently unmatched by either FTA filter-dried blood or oral fluid.

Estrogen regulation of uterine genes in vivo detected by complementary DNA array.

PubMed

Andrade, P M; Silva, I D C G; Borra, R C; de Lima, G R; Baracat, E C

2002-05-01

In the present study, our aim was to identify differentially expressed genes involved in estrogen actions at the endometrium level in rats. Thirty adult rats were ovariectomized four days prior to drug administration for 48 days. Rats were divided in 2 groups: I, control and II, conjugated equine estrogens (CCE). Total RNA was isolated from uterus, and differential expression was analyzed by array technology and RT-PCR. A total of 32 candidate genes were shown to be upregulated or downregulated in groups I or II. Among them, differential expression was already confirmed by RT-PCR for IGFBP5, S12, c-kit, and VEGF, genes whose expression was up regulated during CCE therapy, and casein kinase II and serine kinase expression was the same level in both groups. We have demonstrated that cDNA array represents a powerful approach to identify key molecules in the estrogens therapy. A number of the candidates reported here should provide new markers that may contribute to the detection of target estrogen receptor. This information may also aid the development of new approaches to therapeutic intervention.

Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

NASA Astrophysics Data System (ADS)

Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

2013-03-01

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

The quantification of spermatozoa by real-time quantitative PCR, spectrophotometry, and spermatophore cap size.

PubMed

Doyle, Jacqueline M; McCormick, Cory R; DeWoody, J Andrew

2011-01-01

Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity. © 2010 Blackwell Publishing Ltd.

Validation of endogenous internal real-time PCR controls in renal tissues.

PubMed

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R; Mrug, Michal

2009-01-01

Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright 2009 S. Karger AG, Basel.

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

PubMed Central

Koloušková, Pavla; Stone, James D.

2017-01-01

Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not. PMID:28817728

Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes.

PubMed

Mahakapuge, T A N; Scheerlinck, J-P Y; Rojas, C A Alvarez; Every, A L; Hagen, J

2016-03-01

With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep. Copyright © 2016. Published by Elsevier B.V.

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

PubMed Central

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults. PMID:26998411

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

PubMed

Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Wu, Yeong-Huey; Li, Tsung-Hsien; Leu, Ming-Yih; Chang, Wen-Been; Yang, Wei Cheng

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Differential Expression of CHL1 Gene during Development of Major Human Cancers

PubMed Central

Senchenko, Vera N.; Krasnov, George S.; Dmitriev, Alexey A.; Kudryavtseva, Anna V.; Anedchenko, Ekaterina A.; Braga, Eleonora A.; Pronina, Irina V.; Kondratieva, Tatiana T.; Ivanov, Sergey V.; Zabarovsky, Eugene R.; Lerman, Michael I.

2011-01-01

Background CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis. Methodology/Principal Findings We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases) – in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression. Conclusions/Significance Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer. PMID:21408220

Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa.

PubMed

Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua; Wang, Hongzhi

2016-01-01

The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT-PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation.

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus

PubMed Central

Zhang, Qi-Lin; Zhu, Qian-Hua; Liao, Xin; Wang, Xiu-Qiang; Chen, Tao; Xu, Han-Ting; Wang, Juan; Yuan, Ming-Long; Chen, Jun-Yuan

2016-01-01

Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus. PMID:27869224

Interpretation of Negative Molecular Test Results in Patients With Suspected or Confirmed Ebola Virus Disease: Report of Two Cases.

PubMed

Edwards, Jeffrey K; Kleine, Christian; Munster, Vincent; Giuliani, Ruggero; Massaquoi, Moses; Sprecher, Armand; Chertow, Daniel S

2015-12-01

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the most sensitive quantitative diagnostic assay for detection of Ebola virus in multiple body fluids. Despite the strengths of this assay, we present 2 cases of Ebola virus disease (EVD) and highlight the potential for false-negative results during the early and late stages of EVD. The first case emphasizes the low negative-predictive value of qRT-PCR during incubation and the early febrile stage of EVD, and the second case emphasizes the potential for false-negative results during recovery and late neurologic complications of EVD. Careful interpretation of test results are needed to guide difficult admission and discharge decisions in suspected or confirmed EVD.

ICG: a wiki-driven knowledgebase of internal control genes for RT-qPCR normalization.

PubMed

Sang, Jian; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Xia, Lin; Zou, Dong; Wang, Fan; Xu, Xingjian; Han, Xiaojiao; Fan, Jinqi; Yang, Ye; Zuo, Wanzhu; Zhang, Yang; Zhao, Wenming; Bao, Yiming; Xiao, Jingfa; Hu, Songnian; Hao, Lili; Zhang, Zhang

2018-01-04

Real-time quantitative PCR (RT-qPCR) has become a widely used method for accurate expression profiling of targeted mRNA and ncRNA. Selection of appropriate internal control genes for RT-qPCR normalization is an elementary prerequisite for reliable expression measurement. Here, we present ICG (http://icg.big.ac.cn), a wiki-driven knowledgebase for community curation of experimentally validated internal control genes as well as their associated experimental conditions. Unlike extant related databases that focus on qPCR primers in model organisms (mainly human and mouse), ICG features harnessing collective intelligence in community integration of internal control genes for a variety of species. Specifically, it integrates a comprehensive collection of more than 750 internal control genes for 73 animals, 115 plants, 12 fungi and 9 bacteria, and incorporates detailed information on recommended application scenarios corresponding to specific experimental conditions, which, collectively, are of great help for researchers to adopt appropriate internal control genes for their own experiments. Taken together, ICG serves as a publicly editable and open-content encyclopaedia of internal control genes and accordingly bears broad utility for reliable RT-qPCR normalization and gene expression characterization in both model and non-model organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

PubMed

Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E

2009-12-01

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

PubMed

Ribeiro, Mariana Antunes; dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

2014-11-01

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.

Short communication: comparing real-time PCR and bacteriological cultures for Streptococcus agalactiae and Staphylococcus aureus in bulk-tank milk samples.

PubMed

Zanardi, G; Caminiti, A; Delle Donne, G; Moroni, P; Santi, A; Galletti, G; Tamba, M; Bolzoni, G; Bertocchi, L

2014-09-01

For more than 30 yr, a control plan for Streptococcus agalactiae and Staphylococcus aureus has been carried out in more than 1,500 dairy herds of the province of Brescia (northern Italy). From 2010 to 2011, the apparent prevalence of Strep. agalactiae has been relatively stable around 10%, but the apparent prevalence of Staph. aureus has been greater than 40% with an increasing trend. The aim of this paper was to estimate and compare the diagnostic accuracy of 3 assays for the detection of Strep. agalactiae and Staph. aureus in bulk-tank milk samples (BTMS) in field conditions. The assays were a qualitative and a quantitative bacteriological culture (BC) for each pathogen and a homemade multiplex real-time PCR (rt-PCR). Because a gold standard was not available, the sensitivities (Se) and specificities (Sp) were evaluated using a Bayesian latent class approach. In 2012 we collected one BTMS from 165 dairy herds that were found positive for Strep. agalactiae in the previous 2-yr campaigns of eradication plan. In most cases, BTMS collected in these herds were positive for Staph. aureus as well, confirming the wide spread of this pathogen. At the same time we also collected composite milk samples from all the 8,624 lactating cows to evaluate the within-herd prevalence of Strep. agalactiae. Streptococcus agalactiae samples were cultured using a selective medium Tallium Kristalviolette Tossin, whereas for Staph. aureus, we used Baird Parker modified medium with added Rabbit Plasma Fibrinogen ISO-Formulation. In parallel, BTMS were tested using the rt-PCR. Regarding Strep. agalactiae, the posterior median of Se and Sp of the 2 BC was similar [qualitative BC: Se=98%, posterior credible interval (95%PCI): 94-100%, and Sp=99%, 95%PCI: 96-100%; quantitative BC: Se=99%, 95%PCI: 96-100%, and Sp=99%, 95%PCI: 95-100%] and higher than those of the rt-PCR (at 40 cycle threshold, Se=92%, 95%PCI: 85-97%; Sp=94%, 95%PCI: 88-98%). Also in case of Staph. aureus, the posterior medians of BC were generally higher than those of rt-PCR. In fact, although the Se of BC was slightly lower (rt-PCR at 40 cycle threshold, median Se=99%, 95%PCI: 97-100%, and qualitative BC, median Se=94%, 95%PCI: 87-99%), the Sp was much higher (rt-PCR at 40 cycle threshold, median Sp=67%, 95%PCI: 38-97%; qualitative BC, median Sp=95%; 95%PCI: 76-100%). Our study confirms that BC and rt-PCR are reliable diagnostic tools to detect Strep. agalactiae and Staph. aureus, and rt-PCR results should be confirmed by BC carried out on BTMS and possibly on composite milk samples. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction.

PubMed

Robinson, S; Follo, M; Haenel, D; Mauler, M; Stallmann, D; Tewari, M; Duerschmied, D; Peter, K; Bode, C; Ahrens, I; Hortmann, M

2018-04-15

micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs. We aimed to demonstrate the improved technical performance and diagnostic potential of ddPCR by measuring micro-RNAs in ST-elevation myocardial infarction (STEMI). A dilution series was performed in duplicate on synthetic Caenorrhabditis elegans-miR-39, comparing quantitative real-time PCR (qRT-PCR) and ddPCR. We used ddPCR and qRT-PCR to quantify the serum levels of miR-21, miR-208a and miR-499 between STEMI patients (n=24) and stable coronary artery disease (CAD) patients (n=20). In STEMI, I/R injury was assessed via measurement of ST-segment resolution. In the dilution series, ddPCR demonstrated superior coefficient of variation (12.1%vs.32.9%) and limit of detection (0.9325 vs.2.425copies/μl). In the patient cohort, ddPCR demonstrated greater differences in miR-21 levels (2190.5 vs. 484.7copies/μl; p=0.0004 for ddPCR and 136.4 vs. 122.8copies/μl; p=0.2273 for qRT-PCR) and in miR-208a (0 vs. 24.1copies/μl, p=0.0013 for ddPCR and 0 vs. 0copies/μl, p=0.0032 for qRT-PCR), with similar differences observed in miR-499 levels (9.4 vs. 81.5copies/μl, p<0.0001 for ddPCR and 0 vs. 19.41copies/μl, p<0.0001 for qRT-PCR). ddPCR also more accurately defined STEMI for all miRNAs (area under the curve (AUC) of 0.8021/0.7740/0.9063 for miR-21/208a/499 with ddPCR vs. AUC of 0.6083/0.6917/0.8417 with qRT-PCR). However, there was no association between miR-21/208a/499 levels and ischemia-reperfusion injury. ddPCR demonstrates superiority in both technical performance and diagnostic potential compared to qRT-PCR. Ultimately, this supports its use as a diagnostic method for quantifying micro-RNAs, particularly in large multi-center trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of Gene Expression in Archival Paraffin-Embedded Tissues

PubMed Central

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C.; Shak, Steven; Kiefer, Michael C.; Esteban, Jose M.; Baker, Joffre B.

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-μm FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests. PMID:14695316

miR-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/β-catenin signaling

PubMed Central

Cao, Fengdi; Zhan, Jialin; Chen, Xufeng; Zhang, Kai; Lai, Renfa; Feng, Zhiqiang

2017-01-01

The canonical Wnt/β-catenin signaling is important in the differentiation of human mesenchymal stem cells into osteoblasts. Accumulating evidence suggests that the expression of β-catenin is, in part, regulated by specific microRNAs (miRNAs). The aim of the present study was to investigate the putative roles of miRNAs in osteoblast differentiation. Polymerase chain reaction (PCR) arrays were used to identify miRNAs that were differentially expressed between differentiated and non-differentiated periodontal ligament stem cells (PDLSCs), and reverse transcription-quantitative PCR (RT-qPCR) was used for validation. Since miR-214 was revealed to be significantly downregulated during PDLSC differentiation, its function was further investigated via silencing and overexpression. In addition, osteogenic differentiation of PDLSCs was evaluated at 10 and 21 days following induction, using Alizarin red staining and RT-qPCR analysis for mRNA expression levels of the osteogenic differentiation markers alkaline phosphatase (ALP), osteocalcin and bone sialoprotein. Furthermore, the potential target genes of miR-214 were investigated using a dual-luciferase reporter assay, RT-qPCR and western blot analysis, whereas a TOPflash/FOPflash reporter plasmid system followed by a luciferase assay was used to examine the effects of miR-214 on Wnt/β-catenin signaling. The present results demonstrated that miR-214 was significantly downregulated during the osteoblastic differentiation of PDLSCs. Notably, its overexpression inhibited PDLSC differentiation, whereas its knockdown promoted PDLSC differentiation, as revealed by alterations in mRNA expression of osteoblast-specific genes and ALP. In addition, miR-214 was demonstrated to directly interact with the 3′-untranslated region of the β-catenin gene CTNNB1, and suppressed Wnt/β-catenin signaling through the inhibition of β-catenin. The results of the present study suggested that miR-214 may participate in the regulation of the Wnt/β-catenin signaling pathway, and may have potential as a candidate target for the development of preventive or therapeutic agents for the treatment of patients with osteogenic disorders. PMID:29152645

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

PubMed Central

Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses. PMID:24927412

Application of a Receptor-Binding Capture Quantitative Reverse Transcription-PCR Assay To Concentrate Human Norovirus from Sewage and To Study the Distribution and Stability of the Virus

PubMed Central

Yang, David; Pan, Liangwen; Mandrell, Robert

2012-01-01

Water is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method and compared it to the existing negatively charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (CT) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV were measured as average CT values of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE-concentrated HuNoV was not protected from RNase I and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 103 genomic copies per liter (gc/liter) and 4.3 × 104 gc/liter, respectively. No GI and <2% GII HuNoV were detected in sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage. PMID:22101044

Topographic Quantification of the Transcorneal Electrical Stimulation (TES)-Induced Protective Effects on N-Methyl-N-Nitrosourea-Treated Retinas.

PubMed

Tao, Ye; Chen, Tao; Liu, Zhong-Yu; Wang, Li-Qiang; Xu, Wei-Wei; Qin, Li-Min; Peng, Guang-Hua; Yi-Fei, Huang

2016-09-01

To quantify the transcorneal electrical stimulation (TES)-induced effects on regional photoreceptors and visual signal pathway of N-methyl-N-nitrosourea (MNU)-treated retinas via topographic measurements. N-methyl-N-nitrosourea-administered mice received TES or sham stimulations and were subsequently subjected to electroretinography (ERG), multielectrode array (MEA), and histologic and immunohistochemistry examinations. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses were also performed to determine the mRNA levels of Bax, Bcl-2, Calpain-2, Caspase-3, brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF). Amplitudes of ERG b-wave in the TES-treated mice were significantly larger than those in the sham controls (P < 0.01). Microelectrode array examination revealed that the photoreceptors in TES-treated retina were efficiently preserved (P < 0.01). Morphologic measurements showed that the central retina region was more consolidated than the other areas in the TES-treated mice. Together with the disproportionate distribution of immunostaining in retinal flat mounts, these findings indicated that different rescuing kinetics existed among regional photoreceptors. Compared with the sham controls, a significantly increased signal-to-noise ratio was also found in the TES-treated mice (TES100: 2.02 ± 1.12; TES200: 4.42 ± 1.51; sham: 0.25 ± 0.13; P < 0.01). Moreover, qRT-PCR measurements suggested that the altered expression of several apoptotic factors and neurotrophic cytokines was correlated with TES-induced protection. Regional photoreceptors in the MNU-administered retinas exhibit different sensitivities to TES. Transcorneal electrical stimulation is capable of ameliorating MNU-induced photoreceptor degeneration and rectifying abnormalities in the inner visual signal pathways.

The Long Noncoding RNA Landscape of the Mouse Eye.

PubMed

Chen, Weiwei; Yang, Shuai; Zhou, Zhonglou; Zhao, Xiaoting; Zhong, Jiayun; Reinach, Peter S; Yan, Dongsheng

2017-12-01

Long noncoding RNAs (lncRNAs) are important regulators of diverse biological functions. However, an extensive in-depth analysis of their expression profile and function in mammalian eyes is still lacking. Here we describe comprehensive landscapes of stage-dependent and tissue-specific lncRNA expression in the mouse eye. Affymetrix transcriptome array profiled lncRNA signatures from six different ocular tissue subsets (i.e., cornea, lens, retina, RPE, choroid, and sclera) in newborn and 8-week-old mice. Quantitative RT-PCR analysis validated array findings. Cis analyses and Gene Ontology (GO) annotation of protein-coding genes adjacent to signature lncRNA loci clarified potential lncRNA roles in maintaining tissue identity and regulating eye maturation during the aforementioned phase. In newborn and 8-week-old mice, we identified 47,332 protein-coding and noncoding gene transcripts. LncRNAs comprise 19,313 of these transcripts annotated in public data banks. During this maturation phase of these six different tissue subsets, more than 1000 lncRNAs expression levels underwent ≥2-fold changes. qRT-PCR analysis confirmed part of the gene microarray analysis results. K-means clustering identified 910 lncRNAs in the P0 groups and 686 lncRNAs in the postnatal 8-week-old groups, suggesting distinct tissue-specific lncRNA clusters. GO analysis of protein-coding genes proximal to lncRNA signatures resolved close correlations with their tissue-specific functional maturation between P0 and 8 weeks of age in the 6 tissue subsets. Characterizating maturational changes in lncRNA expression patterns as well as tissue-specific lncRNA signatures in six ocular tissues suggest important contributions made by lncRNA to the control of developmental processes in the mouse eye.

Technique for quantitative RT-PCR analysis directly from single muscle fibers.

PubMed

Wacker, Michael J; Tehel, Michelle M; Gallagher, Philip M

2008-07-01

The use of single-cell quantitative RT-PCR has greatly aided the study of gene expression in fields such as muscle physiology. For this study, we hypothesized that single muscle fibers from a biopsy can be placed directly into the reverse transcription buffer and that gene expression data can be obtained without having to first extract the RNA. To test this hypothesis, biopsies were taken from the vastus lateralis of five male subjects. Single muscle fibers were isolated and underwent RNA isolation (technique 1) or placed directly into reverse transcription buffer (technique 2). After cDNA conversion, individual fiber cDNA was pooled and quantitative PCR was performed using primer-probes for beta(2)-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, insulin-like growth factor I receptor, and glucose transporter subtype 4. The no RNA extraction method provided similar quantitative PCR data as that of the RNA extraction method. A third technique was also tested in which we used one-quarter of an individual fiber's cDNA for PCR (not pooled) and the average coefficient of variation between fibers was <8% (cycle threshold value) for all genes studied. The no RNA extraction technique was tested on isolated muscle fibers using a gene known to increase after exercise (pyruvate dehydrogenase kinase 4). We observed a 13.9-fold change in expression after resistance exercise, which is consistent with what has been previously observed. These results demonstrate a successful method for gene expression analysis directly from single muscle fibers.

Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti.

PubMed

Richardson, Jason; Molina-Cruz, Alvaro; Salazar, Ma Isabel; Black, William

2006-01-01

Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2-3 log(10). Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

Immunohistochemistry reliably detects ALK rearrangements in patients with advanced non-small-cell lung cancer.

PubMed

Han, Xiao-Hong; Zhang, Ning-Ning; Ma, Li; Lin, Dong-Mei; Hao, Xue-Zhi; Liu, Yu-Tao; Wang, Lin; Liu, Peng; Yuan, Zheng; Li, Dan; Lin, Hua; Sun, Yan; Shi, Yuan-Kai

2013-10-01

Accurate determination of anaplastic lymphoma kinase (ALK) rearrangements is critical in identifying ALK-positive patients for targeted therapy in non-small-cell lung cancer (NSCLC). Fluorescence in situ hybridization (FISH) is the current standard method to detect ALK rearrangements but is technically challenging and costly. We compared optimised immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization techniques in this study of 139 samples of advanced NSCLC with non-squamous histology. ALK alteration was found in 32.6 % (43/132) of patients by FISH, 32.9 % (45/137) of patients by IHC and 27.9 % (34/122) of samples by qRT-PCR (concordance rate of 96.9 % between FISH and IHC, 95.7 % between FISH and qRT-PCR, P < 0.001). IHC sensitivity and specificity were 97.7 % and 96.6 %, respectively, while the sensitivity and specificity of qRT-PCR were 89.2 % and 98.7 %, respectively. ALK rearrangements were more common in young patients (P = 0.007), non-smokers or light smokers (P = 0.008) and adenocarcinoma histology, especially with signet ring cell features (P < 0.001). Optimised IHC could be a useful method in screening ALK rearrangements in clinical practice with qRT-PCR as an alternative diagnostic tool to clarify specific ALK variants.

A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

PubMed

Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

2016-06-01

Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti.

PubMed

Dash, Paban Kumar; Boutonnier, Alain; Prina, Eric; Sharma, Shashi; Reiter, Paul

2012-01-22

Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

PubMed

Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system.

PubMed

Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, Martine; Henquell, Cécile; Bailly, Jean-Luc; Peigue-Lafeuille, Hélène; Archimbaud, Christine

2012-10-01

Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections. Copyright © 2012 Elsevier B.V. All rights reserved.

Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

PubMed

Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

2017-01-01

A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G 1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G 1 phase arrest. These results suggest that PANDA promotes G 1 -S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

PubMed

Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

2015-12-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

PubMed Central

Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I.; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J.; Baker, Stephen

2013-01-01

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

PubMed

Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8-100% sensitivity, 100% specificity, 86-89% efficiency and a limit of detection of 12-400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82-90% efficiency and limit of detection of 120-4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PubMed Central

Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains. PMID:26839745

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing.

PubMed

Borgmästars, Emmy; Jazi, Mehrdad Mousavi; Persson, Sofia; Jansson, Linda; Rådström, Peter; Simonsson, Magnus; Hedman, Johannes; Eriksson, Ronnie

2017-12-01

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C q shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification.

PubMed

Wang, Xuan; Yin, Fenggui; Bi, Yuhai; Cheng, Gong; Li, Jing; Hou, Lidan; Li, Yunlong; Yang, Baozhi; Liu, Wenjun; Yang, Limin

2016-12-01

Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Overcoming Heparin-Associated RT-qPCR Inhibition and Normalization Issues for microRNA Quantification in Patients with Acute Myocardial Infarction.

PubMed

Coelho-Lima, Jose; Mohammed, Ashfaq; Cormack, Suzanne; Jones, Samuel; Das, Rajiv; Egred, Mohaned; Panahi, Pedram; Ali, Simi; Spyridopoulos, Ioakim

2018-06-11

 Cardiac-enriched micro ribonucleic acids (miRNAs) are released into the circulation following ST-elevation myocardial infarction (STEMI). Lack of standardized approaches for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) data normalization and presence of RT-qPCR inhibitors (e.g. heparin) in patient blood samples have prevented reproducible miRNA quantification in this cohort and subsequent translation of these biomarkers to clinical practice.  Using a RT-qPCR miRNA screening platform, we identified and validated an endogenous circulating miRNA as a normalization control. In addition, we assessed the effects of in vivo and in vitro anticoagulant drugs administration (heparin and bivalirudin) on three RT-qPCR normalization strategies (global miRNA mean, exogenous spike-in control [cel-miR-39] and endogenous miRNA control). Finally, we evaluated the effect of heparin and its in vitro inhibition with heparinase on the quantification of cardiac-enriched miRNAs in STEMI patients.  miR-425-5p was validated as an endogenous miRNA control. Heparin administration in vitro and in vivo inhibited all RT-qPCR normalization strategies. In contrast, bivalirudin had no effects on cel-miR-39 or miR-425-5p quantification. In vitro RNA sample treatment with 0.3 U of heparinase overcame heparin-induced over-estimation of cardiac-enriched miRNA levels and improved their correlation with high-sensitivity troponin T.  miRNA quantification in STEMI patients receiving heparin is jeopardized by its effect on all RT-qPCR normalization approaches. Use of samples from bivalirudin-treated patients or in vitro treatment of heparin-contaminated samples with heparinase are suitable alternatives for miRNA quantification in this cohort. Finally, we reinforce the evidence that cardiac-enriched miRNAs early after myocardial reperfusion reflect the severity of cardiac injury. Schattauer GmbH Stuttgart.

Direct quantitative detection for cell-free miR-155 in urine: a potential role in diagnosis and prognosis for non-muscle invasive bladder cancer.

PubMed

Zhang, Xin; Zhang, Yanli; Liu, Xinfeng; Fang, Aiju; Wang, Jinfeng; Yang, Yongmei; Wang, Lili; Du, Lutao; Wang, Chuanxin

2016-01-19

High recurrence rates of non-muscle invasive bladder cancer (NMIBC) in patients require lifelong testing and monitoring. The aim of this study is to develop a simplified RT-qPCR method (RT-qPCR-D) which directly quantifies cell-free miR-155 in urine without RNA extraction, and assess it as a potential tool in NMIBC detection. A pilot study including 60 urine samples was used to investigate the feasibility of RT-qPCR-D in detecting cell-free miR-155. Then, miR-155 levels were quantified in a large independent cohort of urine from 162 NIMBC patients, 76 cystitis patients, and 86 healthy donors using the RT-qPCR-D method. Changes of cell-free miR-155 before and after operation were also analyzed in 32 NIMBC patients. In pilot study, we found a significant linear association between RT-qPCR and RT-qPCR-D in urinary miR-155 detection. Both methods showed cell-free miR-155 were significantly increased in NMIBC patients, and could reflect their expression in tissues. Then, the increased expression of cell-free miR-155 was successfully validated in 162 NIMBC patients when compared with cystitis patients and healthy donors. Moreover, it distinguished NMIBC patients from others with 80.2% sensitivity and 84.6% specificity, which was superior to urine cytology. Cell-free miR-155 correlated with NMIBC stage and grade, and was an independent factor for predicting recurrence and progression to muscle invasion. In addition, cell-free miR-155 was significantly decreased after NMIBC patients underwent transurethral bladder resection. In conclusion, detection of cell-free miR-155 in urine using RT-qPCR-D is a simple and noninvasive approach which may be used for NMIBC diagnosis and prognosis prediction.

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

PubMed

Kristof, Jessica; Sakrison, Kellen; Jin, Xiaoping; Nakamaru, Kenji; Schneider, Matthias; Beckman, Robert A; Freeman, Daniel; Spittle, Cindy; Feng, Wenqin

2017-01-01

In preclinical studies, heregulin ( HRG ) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8 , and EIF2B1 ), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

Changes in the Levels of Cryspovirus During In Vitro Development of Cryptosporidium parvum

USDA-ARS?s Scientific Manuscript database

The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of cryspovirus, the viral symbiont of Cryptosporidium parvum, during in vitro development of the protozoan. Cultures of human carcinoma cells (HCT-8) were inoculated with excysting C...

Validation of reference genes for gene expression studies in soybean aphid, Aphis glycines Matsumura

USDA-ARS?s Scientific Manuscript database

Quantitative real-time PCR (qRT-PCR) is a common tool for quantifying mRNA transcripts. To normalize results, a reference gene is mandatory. Aphis glycines is a significant soybean pest, yet gene expression and functional genomics studies are hindered by a lack of stable reference genes. We evalu...

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

PubMed Central

Chao, Jinquan; Yang, Shuguang; Chen, Yueyi; Tian, Wei-Min

2016-01-01

Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR. PMID:27524995

Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

PubMed

Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

2013-08-01

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser. Copyright © 2013 Elsevier Ltd. All rights reserved.

Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

PubMed Central

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R.; Mrug, Michal

2009-01-01

Background Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. Methods To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan® RT-PCR analyses and Affymetrix GeneChip® arrays, were normalized and tested for overall variance and equivalence of the means. Results Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. Conclusion A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. PMID:19729889

Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.

PubMed

Zhang, Fan; Wang, Zhuo-min; Liu, Hong-yu; Bai, Yun; Wei, Sen; Li, Ying; Wang, Min; Chen, Jun; Zhou, Qing-hua

2010-01-01

To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases

PubMed Central

Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

2016-01-01

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer’s disease (AD), Parkinson’s disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies. PMID:27853238

Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases.

PubMed

Rydbirk, Rasmus; Folke, Jonas; Winge, Kristian; Aznar, Susana; Pakkenberg, Bente; Brudek, Tomasz

2016-11-17

Evaluation of gene expression levels by reverse transcription quantitative real-time PCR (RT-qPCR) has for many years been the favourite approach for discovering disease-associated alterations. Normalization of results to stably expressed reference genes (RGs) is pivotal to obtain reliable results. This is especially important in relation to neurodegenerative diseases where disease-related structural changes may affect the most commonly used RGs. We analysed 15 candidate RGs in 98 brain samples from two brain regions from Alzheimer's disease (AD), Parkinson's disease (PD), Multiple System Atrophy, and Progressive Supranuclear Palsy patients. Using RefFinder, a web-based tool for evaluating RG stability, we identified the most stable RGs to be UBE2D2, CYC1, and RPL13 which we recommend for future RT-qPCR studies on human brain tissue from these patients. None of the investigated genes were affected by experimental variables such as RIN, PMI, or age. Findings were further validated by expression analyses of a target gene GSK3B, known to be affected by AD and PD. We obtained high variations in GSK3B levels when contrasting the results using different sets of common RG underlining the importance of a priori validation of RGs for RT-qPCR studies.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

PubMed

Jacob, Tiago R; Peres, Nalu T A; Persinoti, Gabriela F; Silva, Larissa G; Mazucato, Mendelson; Rossi, Antonio; Martinez-Rossi, Nilce M

2012-05-01

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

PubMed

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

PubMed

Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

2011-07-01

Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 Subtype C infected patients in Northern India

PubMed Central

Agarwal, Atima; Sankaran, Sumathi; Vajpayee, Madhu; Sreenivas, V; Seth, Pradeep; Dandekar, Satya

2014-01-01

Background Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. Objectives The objective of this study was to develop and validate an affordable; one step Real-Time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. Results We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. Conclusions The RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India. PMID:17962068

Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

PubMed Central

Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects. PMID:24466124

Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR.

PubMed

Theis, Torsten; Skurray, Ronald A; Brown, Melissa H

2007-08-01

Quantitative real-time PCR (qRT-PCR) has become a routine technique for gene expression analysis. Housekeeping genes are customarily used as endogenous references for the relative quantification of genes of interest. The aim of this study was to develop a quantitative real-time PCR assay to analyze gene expression in multidrug resistant Staphylococcus aureus in the presence of cationic lipophilic substrates of multidrug transport proteins. Eleven different housekeeping genes were analyzed for their expression stability in the presence of a range of concentrations of four structurally different antimicrobial compounds. This analysis demonstrated that the genes rho, pyk and proC were least affected by rhodamine 6G and crystal violet, whereas fabD, tpiA and gyrA or fabD, proC and pyk were stably expressed in cultures grown in the presence of ethidium or berberine, respectively. Subsequently, these housekeeping genes were used as internal controls to analyze expression of the multidrug transport protein QacA and its transcriptional regulator QacR in the presence of the aforementioned compounds. Expression of qacA was induced by all four compounds, whereas qacR expression was found to be unaffected, reduced or enhanced. This study demonstrates that staphylococcal gene expression, including housekeeping genes previously used to normalize qRT-PCR data, is affected by growth in the presence of different antimicrobial compounds. Thus, identification of suitable genes usable as a control set requires rigorous testing. Identification of a such a set enabled them to be utilized as internal standards for accurate quantification of transcripts of the qac multidrug resistance system from S. aureus grown under different inducing conditions. Moreover, the qRT-PCR assay presented in this study may also be applied to gene expression studies of other multidrug transporters from S. aureus.

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

PubMed

Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Diagnosis of cytomegalovirus pneumonia by quantitative polymerase chain reaction using bronchial washing fluid from patients with hematologic malignancies

PubMed Central

Choi, Joon Young; Lee, Hea Yon; Lee, Jong Wook; Lee, Dong Gun

2017-01-01

Background The incidence of cytomegalovirus (CMV) pneumonia is increasing in patients diagnosed with hematologic malignancies. The utility of CMV-DNA viral load measurement has not been standardized, and viral cut-off values have not been established. This study was designed to investigate the utility of CMV quantitative real-time PCR (qRT-PCR) using bronchial washing fluid. Methods We retrospectively reviewed the microbiologic and pathologic results of bronchial washing fluid and biopsy specimens in addition to the patients' clinical characteristics. Results A total of 565 CMV qRT-PCR assays were performed using bronchial washing fluid from patients with hematologic malignancies. Among them, 101 were positive for CMV by qRT-PCR; of these, 24 were diagnosed with CMV pneumonia and 70 with CMV infection, and 7 were excluded due to a diagnosis of invasive pulmonary aspergillosis rather than viral pneumonia. The median CMV load determined by qPCR was 1.8 × 105 copies/mL (3.6 103-1.5 × 108) in CMV pneumonia patients and 3.0 × 103 copies/mL (5.0 × 102-1.1 × 105) in those diagnosed with CMV infection (P < 0.01). Using the ROC curve, the optimal inflection points were 18,900 copies/mL (137,970 IU/mL) in post-bone marrow transplantation (BMT) patients, 316,415 copies/mL (2,309,825 IU/mL) in no-BMT patients and 28,774 copies/mL (210,054 IU/mL) in all patients. Conclusions The CMV titers in bronchial washing fluid determined by qRT-PCR differed significantly between patients diagnosed with CMV pneumonia and those with CMV infection. The viral cut-off values in bronchial washing fluid were suggested for the diagnosis of CMV pneumonia, which were different depending on the BMT status. PMID:28061469

Diagnosis of cytomegalovirus pneumonia by quantitative polymerase chain reaction using bronchial washing fluid from patients with hematologic malignancies.

PubMed

Lee, Hwa Young; Rhee, Chin Kook; Choi, Joon Young; Lee, Hea Yon; Lee, Jong Wook; Lee, Dong Gun

2017-06-13

The incidence of cytomegalovirus (CMV) pneumonia is increasing in patients diagnosed with hematologic malignancies. The utility of CMV-DNA viral load measurement has not been standardized, and viral cut-off values have not been established. This study was designed to investigate the utility of CMV quantitative real-time PCR (qRT-PCR) using bronchial washing fluid. We retrospectively reviewed the microbiologic and pathologic results of bronchial washing fluid and biopsy specimens in addition to the patients' clinical characteristics. A total of 565 CMV qRT-PCR assays were performed using bronchial washing fluid from patients with hematologic malignancies. Among them, 101 were positive for CMV by qRT-PCR; of these, 24 were diagnosed with CMV pneumonia and 70 with CMV infection, and 7 were excluded due to a diagnosis of invasive pulmonary aspergillosis rather than viral pneumonia. The median CMV load determined by qPCR was 1.8 × 105 copies/mL (3.6 103-1.5 × 108) in CMV pneumonia patients and 3.0 × 103 copies/mL (5.0 × 102-1.1 × 105) in those diagnosed with CMV infection (P < 0.01). Using the ROC curve, the optimal inflection points were 18,900 copies/mL (137,970 IU/mL) in post-bone marrow transplantation (BMT) patients, 316,415 copies/mL (2,309,825 IU/mL) in no-BMT patients and 28,774 copies/mL (210,054 IU/mL) in all patients. The CMV titers in bronchial washing fluid determined by qRT-PCR differed significantly between patients diagnosed with CMV pneumonia and those with CMV infection. The viral cut-off values in bronchial washing fluid were suggested for the diagnosis of CMV pneumonia, which were different depending on the BMT status.

Altered expression of four miRNA (miR-1238-3p, miR-202-3p, miR-630 and miR-766-3p) and their potential targets in peripheral blood from vitiligo patients.

PubMed

Shang, Zhiwei; Li, Hongwen

2017-10-01

Vitiligo is an acquired skin disease with pigmentary disorder. Autoimmune destruction of melanocytes is thought to be major factor in the etiology of vitiligo. miRNA-based regulators of gene expression have been reported to play crucial roles in autoimmune disease. Therefore, we attempt to profile the miRNA expressions and predict their potential targets, assessing the biological functions of differentially expressed miRNA. Total RNA was extracted from peripheral blood of vitiligo (experimental group, n = 5) and non-vitiligo (control group, n = 5) age-matched patients. Samples were hybridized to a miRNA array. Box, scatter and principal component analysis plots were performed, followed by unsupervised hierarchical clustering analysis to classify the samples. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted for validation of microarray data. Three different databases, TargetScan, PITA and microRNA.org, were used to predict the potential target genes. Gene ontology (GO) annotation and pathway analysis were performed to assess the potential functions of predicted genes of identified miRNA. A total of 100 (29 upregulated and 71 downregulated) miRNA were filtered by volcano plot analysis. Four miRNA were validated by quantitative RT-PCR as significantly downregulated in the vitiligo group. The functions of predicted target genes associated with differentially expressed miRNA were assessed by GO analysis, showing that the GO term with most significantly enriched target genes was axon guidance, and that the axon guidance pathway was most significantly correlated with these miRNA. In conclusion, we identified four downregulated miRNA in vitiligo and assessed the potential functions of target genes related to these differentially expressed miRNA. © 2017 Japanese Dermatological Association.

The ontogenic expressions of multiple vesicular glutamate transporters during postnatal development of rat pineal gland.

PubMed

Yoshida, S; Ina, A; Konno, J; Wu, T; Shutoh, F; Nogami, H; Hisano, S

2008-03-18

The pineal gland expresses vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2), which are thought to transport glutamate into synaptic-like microvesicles in the pinealocytes. Recently, we reported that the rat pineal gland also expresses VGLUT1v which is a novel variant of VGLUT1 during the perinatal period. To explore the biological significance of these VGLUT expressions in pineal development, we studied the ontogeny of VGLUT in this gland by in situ hybridization, immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR) using rats. Histological analysis revealed that intensities of VGLUT1 hybridization signal and immunostaining drastically increase by postnatal day (P) 7, whereas VGLUT2 expression exhibits high levels of mRNA and protein at birth and decreases gradually from P7 onward. Quantitative RT-PCR analysis supported these histological observations, showing that expressions of VGLUT1 and VGLUT2 exhibit opposite patterns to each other. Coinciding with VGLUT1-upregulation, RT-PCR data showed that expressions of dynamin 1 and endophilin 1, which are factors predictably involved in the endocytotic recovery of VGLUT1-associated vesicle, are also increased by P7. Quantitative RT-PCR analysis of VGLUT1v demonstrated that its mRNA expression is upregulated by P7, kept at the same level until P14, and apparently decreased at P21, suggesting its functional property required for a certain developmental event. Moreover, a comparison of mRNA expressions at daytime and nighttime revealed that neither VGLUT1 nor VGLUT1v shows any difference in both P7 and P21 glands, whereas VGLUT2 is significantly lower at daytime than at nighttime at P21 but not P7, the time point at which the melatonin rhythm is not yet generated. The present study shows that expressions of these VGLUT types are differentially regulated during postnatal pineal development, each presumably participating in physiologically distinct glutamatergic functions.

Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR.

PubMed

Sajitha, K L; Dev, Suma Arun; Maria Florence, E J

2016-07-01

Bacillus subtilis is a potent biocontrol agent producing a wide array of antifungal lipopeptides for the inhibition of fungal growth. B. subtilis B1 isolated from market-available compost provided an efficient control of rubberwood sapstain fungus, Lasiodiplodia theobromae. The current study is aimed to identify and characterize the lipopeptides responsible for the biocontrol of rubberwood sapstain fungus by Bacillus subtilis B1. The bacterial whole-cell surface extract from the dual culture of B. subtilis B1 and sapstain fungus (L. theobromae) was analysed using MALDI-TOF-MS. The protonated as well as sodium, potassium adducts of homologues of iturin C, surfactin, bacillomycin D and fengycin A and B were identified and expression of the lipopeptide biosynthetic genes could be confirmed through RT-PCR. This is the first report of mycobacillin and trimethylsilyl derivative of bacilysin during antagonism through MALDI-TOF-MS. MALDI-TOF-MS with RT-PCR offered easy platforms to characterize the antifungal lipopeptides. The identification of antifungal lipopeptides can lead to the formulation of prospective biocontrol by-products which have wide-scale utility.

MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer.

PubMed

Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels; Sørensen, Flemming Brandt; Jakobsen, Anders; Hansen, Torben Frøstrup

2016-01-01

MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer. We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa. From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation). We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.

Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti

PubMed Central

2012-01-01

Background Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. Results We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). Conclusions The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods. PMID:22264275

Ultrasensitive detection of oncogenic human papillomavirus in oropharyngeal tissue swabs.

PubMed

Isaac, Andre; Kostiuk, Morris; Zhang, Han; Lindsay, Cameron; Makki, Fawaz; O'Connell, Daniel A; Harris, Jeffrey R; Cote, David W J; Seikaly, Hadi; Biron, Vincent L

2017-01-14

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) caused by oncogenic human papillomavirus (HPV) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV E6 and E7 oncoproteins or by p16 immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as an ultra-sensitive and highly precise method of nucleic acid quantification for biomarker analysis. To validate the use of a minimally invasive assay for detection of oncogenic HPV based on oropharyngeal swabs using ddPCR. Secondary objectives were to compare the accuracy of ddPCR swabs to fresh tissue p16 IHC and RT-qPCR, and to compare the cost of ddPCR with p16 IHC. We prospectively included patients with p16 + oral cavity/oropharyngeal cancer (OC/OPSCC), and two control groups: p16 - OC/OPSCC patients, and healthy controls undergoing tonsillectomy. All underwent an oropharyngeal swab with ddPCR for quantitative detection of E6 and E7 mRNA. Surgical specimens had p16 IHC performed. Agreement between ddPCR and p16 IHC was determined for patients with p16 positive and negative OC/OPSCC as well as for healthy control patients. The sensitivity and specificity of ddPCR of oropharyngeal swabs were calculated against p16 IHC for OPSCC. 122 patients were included: 36 patients with p16 + OPSCC, 16 patients with p16 - OPSCC, 4 patients with p16 + OCSCC, 41 patients with p16 - OCSCC, and 25 healthy controls. The sensitivity and specificity of ddPCR of oropharyngeal swabs against p16 IHC were 92 and 98% respectively, using 20-50 times less RNA than that required for conventional RT-qPCR. Overall agreement between ddPCR of tissue swabs and p16 of tumor tissue was high at ĸ = 0.826 [0.662-0.989]. Oropharyngeal swabs analyzed by ddPCR is a quantitative, rapid, and effective method for minimally invasive oncogenic HPV detection. This assay represents the most sensitive and accurate mode of HPV detection in OPSCC without a tissue biopsy in the available literature.

Correlative mRNA and Protein Expression of Middle and Inner Ear Inflammatory Cytokines during Mouse Acute Otitis Media

PubMed Central

Trune, Dennis R.; Kempton, Beth; Hausman, Frances A.; Larrain, Barbara E.; MacArthur, Carol J.

2015-01-01

Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 hours. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 hour samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2 to 122 fold higher at 18 hours, declining slightly from there at 24 hours. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media. PMID:25922207

Correlative mRNA and protein expression of middle and inner ear inflammatory cytokines during mouse acute otitis media.

PubMed

Trune, Dennis R; Kempton, Beth; Hausman, Frances A; Larrain, Barbara E; MacArthur, Carol J

2015-08-01

Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 h. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1β (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 h samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2-122 fold higher at 18 h, declining slightly from there at 24 h. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcriptome Analysis of the Dihydrotestosterone-Exposed Fetal Rat Gubernaculum Identifies Common Androgen and Insulin-Like 3 Targets1

PubMed Central

Barthold, Julia S.; Wang, Yanping; Robbins, Alan; Pike, Jack; McDowell, Erin; Johnson, Kamin J.; McCahan, Suzanne M.

2013-01-01

ABSTRACT Androgens and insulin-like 3 (INSL3) are required for development of the fetal gubernaculum and testicular descent. Previous studies suggested that the INSL3-exposed fetal gubernacular transcriptome is enriched for genes involved in neural pathways. In the present study, we profiled the transcriptome of fetal gubernaculum explants exposed to dihydrotestosterone (DHT) and compared this response to that with INSL3. We exposed fetal (Embryonic Day 17) rat gubernacula to DHT for 24 h (10 and 30 nM) or 6 h (1 and 10 nM) in organ culture and analyzed gene expression relative to that of vehicle-treated controls using Affymetrix arrays. Results were annotated using functional, pathway, and promoter analyses and independently validated for selected transcripts using quantitative RT-PCR (qRT-PCR). Transcripts were differentially expressed after 24 h but not 6 h. Most highly overrepresented functional categories included those related to gene expression, skeletal and muscular development and function, and Wnt signaling. Promoter response elements enriched in the DHT-specific transcriptome included consensus sequences for c-ETS1, ELK1, CREB, CRE-BP1/c-June, NRF2, and USF. We observed that 55% of DHT probe sets were also differentially expressed after INSL3 exposure and that the direction of change was the same in 96%. The qRT-PCR results confirmed that DHT increased expression of the INSL3-responsive genes Crlf1 and Chrdl2 but reduced expression of Wnt4. We also validated reduced Tgfb2 and Cxcl12 and increased Slit3 expression following DHT exposure. These data suggest a robust overlap in the DHT- and INSL3-regulated transcriptome that may be mediated in part by CREB signaling and a common Wnt pathway response for both hormones in the fetal gubernaculum. PMID:24174575

Assessment of Phthalate-induced Changes in Fetal Rat Testis Gene Expression using an rt-PCR Drug Metabolism Array.

EPA Science Inventory

Phthalate esters are a large family of compounds used in many industrial and commercial products. Based on numerous studies, phthalates such as diethyl hexyl phthalate (DEHP) produce reproductive malformations in male rodents through reduction of testosterone production and gene ...

WetLab-2: Providing Quantitative PCR Capabilities on ISS

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy Kar Chuen; Almeida, Eduardo; Boone, Travis David; Schonfeld, Julie; Tran, Luan Hoang

2015-01-01

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a system capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project has developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage that it uses non-toxic chemicals, alcohols or other organics. The resulting RNA is transferred into a pipette and then dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. These reaction tubes are mounted on rotors to centrifuge the liquid to the reaction window of the tube using a cordless drill. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The resulting process takes less than 30 min to have tubes ready for loading into the qRT-PCR unit.The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, the Cepheid SmartCycler, that will fly in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid thermal ramp times and four-color detection. The ability to detect up to four fluorescent channels will enable multiplex assays that can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system will have the capability to downlink data from the ISS to the ground after a completed run and to uplink new programs. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time for subsequent trials, without the need for sample return and re-flight to sample multigenerational changes. The system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015. The WetLab-2 Project is supported by NASAs ISS Program at JSC, Code OZ.

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

PubMed

Wang, Hye-Young; Ahn, Sungwoo; Park, Sunyoung; Kim, SeungIl; Lee, Hyeyoung

2017-01-01

Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression. © 2016 S. Karger AG, Basel.

Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

PubMed Central

Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A.; Liu, Jie; Kirkpatrick, Beth D.; Chowdhury, Anwarul H.; Jamil, Khondoker M.; Haque, Rashidul; Petri, William A.; Houpt, Eric R.

2014-01-01

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture+/qPCR+ and culture−/qPCR+ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

Gene expression profiling in patients with polymyalgia rheumatica before and after symptom-abolishing glucocorticoid treatment.

PubMed

Kreiner, Frederik Flindt; Borup, Rehannah; Nielsen, Finn Cilius; Schjerling, Peter; Galbo, Henrik

2017-08-07

The pathophysiology, including the impact of gene expression, of polymyalgia rheumatica (PMR) remains elusive. We profiled the gene expression in muscle tissue in PMR patients before and after glucocorticoid treatment. Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 arrays in muscle biopsies from 8 glucocorticoid-naive patients with PMR and 10 controls before and after prednisolone-treatment for 14 days. For 14 genes, quantitative real-time PCR (qRT-PCR, n = 9 in both groups) was used to validate the microarray findings and to further investigate the expression of genes of particular interest. Prednisolone normalized erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in PMR patients. A total of 165 putatively clinically relevant, differentially expressed genes were identified (cut-off: fold difference > ±1.2, difference of mean > 30, and p < 0.05); of these, 78 genes differed between patients and controls before treatment, 131 genes responded to treatment in a given direction only in patients, and 44 fulfilled both these criteria. In 43 of the 44 genes, treatment counteracted the initial difference. Functional clustering identified themes of biological function, including regulation of protein biosynthesis, and regulation of transcription and of extracellular matrix processes. Overall, qRT-PCR confirmed the microarray findings: Microarray-detected group differences were confirmed for 9 genes in 17 of 18 comparisons (same magnitude and direction of change); lack of group differences in microarray testing was confirmed for 5 genes in 8 of 10 comparisons. Before treatment, using qRT-PCR, expression of interleukin 6 (IL-6) was found to be 4-fold higher in patients (p < 0.05). This study identifies genes in muscle, the expression of which may impact the pathophysiology of PMR. Moreover, the study adds further evidence of the importance of IL-6 in the disease. Follow-up studies are needed to establish the exact pathophysiological relevance of the identified genes. The study was retrospectively listed on the ISRCTN registry with study ID ISRCTN69503018 and date of registration the 26th of July 2017.

Establishment and validation of analytical reference panels for the standardization of quantitative BCR-ABL1 measurements on the international scale.

PubMed

White, Helen E; Hedges, John; Bendit, Israel; Branford, Susan; Colomer, Dolors; Hochhaus, Andreas; Hughes, Timothy; Kamel-Reid, Suzanne; Kim, Dong-Wook; Modur, Vijay; Müller, Martin C; Pagnano, Katia B; Pane, Fabrizio; Radich, Jerry; Cross, Nicholas C P; Labourier, Emmanuel

2013-06-01

Current guidelines for managing Philadelphia-positive chronic myeloid leukemia include monitoring the expression of the BCR-ABL1 (breakpoint cluster region/c-abl oncogene 1, non-receptor tyrosine kinase) fusion gene by quantitative reverse-transcription PCR (RT-qPCR). Our goal was to establish and validate reference panels to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate global standardization on the international scale (IS). Four-level secondary reference panels were manufactured under controlled and validated processes with synthetic Armored RNA Quant molecules (Asuragen) calibrated to reference standards from the WHO and the NIST. Performance was evaluated in IS reference laboratories and with non-IS-standardized RT-qPCR methods. For most methods, percent ratios for BCR-ABL1 e13a2 and e14a2 relative to ABL1 or BCR were robust at 4 different levels and linear over 3 logarithms, from 10% to 0.01% on the IS. The intraassay and interassay imprecision was <2-fold overall. Performance was stable across 3 consecutive lots, in multiple laboratories, and over a period of 18 months to date. International field trials demonstrated the commutability of the reagents and their accurate alignment to the IS within the intra- and interlaboratory imprecision of IS-standardized methods. The synthetic calibrator panels are robust, reproducibly manufactured, analytically calibrated to the WHO primary standards, and compatible with most BCR-ABL1 RT-qPCR assay designs. The broad availability of secondary reference reagents will further facilitate interlaboratory comparative studies and independent quality assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results and the optimization of current and new therapeutic approaches for chronic myeloid leukemia. © 2013 American Association for Clinical Chemistry.

Arabidopsis gene expression patterns during spaceflight

NASA Astrophysics Data System (ADS)

Paul, A.-L.; Ferl, R. J.

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

Method for measuring the size distribution of airborne rhinovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, M.L.; Goth-Goldstein, R.; Apte, M.G.

About 50% of viral-induced respiratory illnesses are caused by the human rhinovirus (HRV). Measurements of the concentrations and sizes of bioaerosols are critical for research on building characteristics, aerosol transport, and mitigation measures. We developed a quantitative reverse transcription-coupled polymerase chain reaction (RT-PCR) assay for HRV and verified that this assay detects HRV in nasal lavage samples. A quantitation standard was used to determine a detection limit of 5 fg of HRV RNA with a linear range over 1000-fold. To measure the size distribution of HRV aerosols, volunteers with a head cold spent two hours in a ventilated research chamber.more » Airborne particles from the chamber were collected using an Andersen Six-Stage Cascade Impactor. Each stage of the impactor was analyzed by quantitative RT-PCR for HRV. For the first two volunteers with confirmed HRV infection, but with mild symptoms, we were unable to detect HRV on any stage of the impactor.« less

Real-Time Quantitative PCR (QPCR) and Reverse Transcription-QPCR for Detection and Enumeration of Total Yeasts in Wine▿

PubMed Central

Hierro, Núria; Esteve-Zarzoso, Braulio; González, Ángel; Mas, Albert; Guillamón, Jose M.

2006-01-01

Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

Therapeutic siRNAs for dominant genetic skin diseases including pachyonychia congenita

PubMed Central

Leachman, Sancy A.; Hickerson, Robyn P.; Hull, Peter R.; Smith, Frances J. D.; Milstone, Leonard M.; Lane, E. Birgitte; Bale, Sherri J.; Roop, Dennis R.; McLean, W. H. Irwin; Kaspar, Roger L.

2008-01-01

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this “first-in-skin” siRNA may herald a paradigm shift in the treatment of dominant negative genetic disorders. PMID:18495438

Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita.

PubMed

Leachman, Sancy A; Hickerson, Robyn P; Hull, Peter R; Smith, Frances J D; Milstone, Leonard M; Lane, E Birgitte; Bale, Sherri J; Roop, Dennis R; McLean, W H Irwin; Kaspar, Roger L

2008-09-01

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this "first-in-skin" siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.

Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.

PubMed

Liu, Jing; Wang, Qun; Sun, Minying; Zhu, Linlin; Yang, Michael; Zhao, Yu

2014-01-01

Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

Structural shifts of fecal microbial communities in rats with acute rejection after liver transplantation.

PubMed

Xie, Yirui; Luo, Zhuanbo; Li, Zhengfeng; Deng, Min; Liu, Hao; Zhu, Biao; Ruan, Bing; Li, Lanjuan

2012-08-01

Bacterial translocation and the development of sepsis after orthotopic liver transplantation (OLT) may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microbiota. This study monitored structural shifts of gut microbiota in rats with OLT using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (RT-qPCR). RT-qPCR targets six major microorganisms (Domain Bacteria, Bacteroides, Bifidobacteria, Enterobacteriaceae, Lactobacillus and Clostridium leptum subgroup). Isograft, Allograft and Sham model were studied. Bacterial translocation to host organs and plasma endotoxin were determined. Alteration in gut microbiota was associated with the elevation of plasma endotoxin and a higher rate of bacterial translocation (BT) to liver in rats with acute rejection. Dynamic analysis of DGGE fingerprints showed that the gut microbiota structure of animals in the three groups was similar before the operation. But significant alterations in the composition of fecal microbiota in Allograft group were observed at 1 and 2 weeks after the OLT. The acute rejection was accompanied by the shifts of gut microbiota towards members of Bacteroides and Ruminococcus. Results from RT-qPCR indicated that Bacteroides significantly increased at 2 weeks after the OLT, whereas numbers of Bifidobacterium spp. decreased at 1 week and recovered at 2 weeks after the OLT. In summary, our data showed that rats with acute rejection after OLT exhibited significant structure shifts in the gut microbiota which dominant by overgrowth of Bacteroides and Ruminococcus, and these were associated with elevation of plasma endotoxin and higher rate of BT.

Development Status of the WetLab-2 Project: New Tools for On-orbit Real-time Quantitative Gene Expression.

NASA Technical Reports Server (NTRS)

Jung, Jimmy; Parra, Macarena P.; Almeida, Eduardo; Boone, Travis; Chinn, Tori; Ricco, Antonio; Souza, Kenneth; Hyde, Liz; Rukhsana, Yousuf; Richey, C. Scott

2013-01-01

The primary objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform to facilitate gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens grown or cultured on orbit. The WetLab-2 equipment will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. In addition to the logistical benefits of in-situ sample processing and analysis, conducting qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms. The system can also validate terrestrial analyses of samples returned from ISS by providing quantitative on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experimental parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Finally, WetLab-2 can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-5 in Aug. 2014.Progress to date: The WetLab-2 project completed a thorough study of commercially available qRT-PCR systems and performed a downselect based on both scientific and engineering requirements. The selected instrument, the Cepheid SmartCycler, has advantages including modular design (16 independent PCR modules), low power consumption, and rapid ramp times. The SmartCycler has multiplex capabilities, assaying up to four genes of interest in each of the 16 modules. The WetLab-2 team is currently working with Cepheid to modify the unit for housing within an EXPRESS rack locker on the ISS. This will enable the downlink of data to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project is currently designing a module that will lyse the cells and extract RNA of sufficient quality for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. Current testing focuses on two promising commercial products and chemistries that allow for RNA extraction with minimal complexity and crew time.

Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.

PubMed

Gao, Mengmeng; Liu, Yaping; Ma, Xiao; Shuai, Qin; Gai, Junyi; Li, Yan

2017-01-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.

An In-Depth Evaluation of the Validity and Logistics Surrounding the Testing of AR-V7 mRNA Expression in Circulating Tumor Cells.

PubMed

Sieuwerts, Anieta M; Mostert, Bianca; van der Vlugt-Daane, Michelle; Kraan, Jaco; Beaufort, Corine M; Van, Mai; Prager, Wendy J C; De Laere, Bram; Beije, Nick; Hamberg, Paul; Westgeest, Hans M; Tascilar, Metin; Dirix, Luc Y; Onstenk, Wendy; de Wit, Ronald; Lolkema, Martijn P; Mathijssen, Ron H J; Martens, John W M; Sleijfer, Stefan

2018-05-01

Recent reports have emphasized the clinical relevance of detecting AR-V7 in circulating tumor cells (CTCs). Our aim was to set up a validated multicenter pipeline to measure AR-V7 by quantitative RT-PCR (RT-qPCR) in RNA isolated from CellSearch-enriched CTCs to provide an AR-V7-positive or AR-V7-negative score in a clinically acceptable time range. CellSearch-enirched CTCs from patients with metastatic castration-resistant prostate cancer were characterized by RT-qPCR. After optimization, it was prospectively tested whether it was possible to report the AR-V7 status within 11 days (PRELUDE study). In the range of the RNA equivalent of 0.2 to 12 VCaP cells, the CV for AR-V7 was 9% (n = 37). The limit of detection was 0.3, and the limit of quantitation was 3 cells in the final RT-qPCR. No differences were observed between AR-V7 data generated by five technicians or in two different laboratories. For the 45 patients in PRELUDE, 13 patients were ineligible, 22 patients were AR-V7 negative, and 10 were AR-V7 positive. The median time to inform the physician of the test result was 7 days (range, 2 to 11 days). This assay can establish the AR-V7 status in CTCs from patients with metastatic castration-resistant prostate cancer. Furthermore, it was possible to provide an AR-V7 outcome within 11 days, indicating that it may be used to choose between an anti-androgen receptor or taxane-based cabazitaxel treatment. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Evaluation of reference genes in mouse preimplantation embryos for gene expression studies using real-time quantitative RT-PCR (RT-qPCR).

PubMed

Jeong, Jae-Kyo; Kang, Min-Hee; Gurunathan, Sangiliyandi; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

2014-09-25

Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) is the most sensitive, and valuable technique for rare mRNA detection. However, the expression profiles of reference genes under different experimental conditions, such as different mouse strains, developmental stage, and culture conditions have been poorly studied. mRNA stability of the actb, gapdh, sdha, ablim, ywhaz, sptbn, h2afz, tgfb1, 18 s and wrnip genes was analyzed. Using the NormFinder program, the most stable genes are as follows: h2afz for the B6D2F-1 and C57BL/6 strains; sptbn for ICR; h2afz for KOSOM and CZB cultures of B6D2F-1 and C57BL/6 strain-derived embryos; wrnip for M16 culture of B6D2F-1 and C57BL/6 strain-derived embryos; ywhaz, tgfb1, 18 s, 18 s, ywhaz, and h2afz for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst embryonic stages cultured in KSOM medium, respectively; h2afz, wrnip, wrnip, h2afz, ywhaz, and ablim for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in CZB medium, respectively; 18 s, h2afz, h2afz, actb, h2afz, and wrnip for zygote, 2-cell, 4-cell, 8-cell, molular, and blastocyst stage embryos cultured in M16 medium, respectively. These results demonstrated that candidate reference genes for normalization of target gene expression using RT-qPCR should be selected according to mouse strains, developmental stage, and culture conditions.

Comprehensive Analysis of CBFβ-MYH11 Fusion Transcripts in Acute Myeloid Leukemia by RT-PCR Analysis

PubMed Central

Kadkol, ShriHari S.; Bruno, Annette; Dodge, Carol; Lindgren, Valerie; Ravandi, Farhad

2004-01-01

CBFβ-MYH11 fusion transcripts are expressed in acute myeloid leukemias of the M4Eo subtype. Patients who express CBFβ-MYH11 fusion transcripts respond favorably to high-dose chemotherapy and are generally spared allogeneic bone marrow transplantation. Hence it is important to identify this fusion in all patients with acute myeloid leukemia M4Eo leukemia. The fusion can be detected by cytogenetics, fluorescence in-situ hybridization (FISH), or by molecular analysis with RT-PCR. Multiple fusion transcripts arising as a result of various breakpoints in the CBFβ and MYH11 have been identified. In this report we describe a comprehensive RT-PCR assay to identify all known fusion transcripts and provide an algorithm for molecular analysis of CBFβ-MYH11 fusions from patient specimens. Further, identification of the fusion transcript by such an assay would help in the diagnosis and follow up of patients with cryptic inversion 16 translocations (such as patient 2 in this report) not detected by standard cytogenetics or FISH and for rational design of probes for quantitative analysis by real-time PCR. PMID:14736823

Development of a Real-Time, TaqMan Reverse Transcription-PCR Assay for Detection and Differentiation of Lyssavirus Genotypes 1, 5, and 6

PubMed Central

Wakeley, P. R.; Johnson, N.; McElhinney, L. M.; Marston, D.; Sawyer, J.; Fooks, A. R.

2005-01-01

Several reverse transcription-PCR (RT-PCR) methods have been reported for the detection of rabies and rabies-related viruses. These methods invariably involve multiple transfers of nucleic acids between different tubes, with the risk of contamination leading to the production of false-positive results. Here we describe a single, closed-tube, nonnested RT-PCR with TaqMan technology that distinguishes between classical rabies virus (genotype 1) and European bat lyssaviruses 1 and 2 (genotypes 5 and 6) in real time. The TaqMan assay is rapid, sensitive, and specific and allows for the genotyping of unknown isolates concomitant with the RT-PCR. The assay can be applied quantitatively and the use of an internal control enables the quality of the isolated template to be assessed. Despite sequence heterogeneity in the N gene between the different genotypes, a universal forward and reverse primer set has been designed, allowing for the simplification of previously described assays. We propose that within a geographically constrained area, this assay will be a useful tool for the detection and differentiation of members of the Lyssavirus genus. PMID:15956398

Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

NASA Technical Reports Server (NTRS)

Tran, L.; Parra, Macarena P.; Jung, J.; Boone, T.; Schonfeld, Julie; Almeida, Eduardo

2017-01-01

The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

Identification of novel and robust internal control genes from Volvariella volvacea that are suitable for RT-qPCR in filamentous fungi.

PubMed

Tao, Yongxin; van Peer, Arend Frans; Huang, Qianhui; Shao, Yanping; Zhang, Lei; Xie, Bin; Jiang, Yuji; Zhu, Jian; Xie, Baogui

2016-07-12

The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR.

Rapid detection of Cronobacter sakazakii by real-time PCR based on the cgcA gene and TaqMan probe with internal amplification control.

PubMed

Hu, Shuangfang; Yu, Yigang; Li, Rong; Wu, Xinwei; Xiao, Xinglong; Wu, Hui

2016-03-01

Cronobacter sakazakii is a severe virulent strain that is frequently detected in powdered infant formula (PIF). Therefore, it is necessary to develop a fast and specific detection method. The specificity of our newly developed quantitative real-time PCR (qRT-PCR) was validated with DNA from 46 strains. Among them, 12 C. sakazakii strains were correctly amplified, whereas no positive florescent signal was observed from 34 nontarget controls. The detection limit of C. sakazakii was about 110 CFU/mL in broth and 1100 CFU/g in PIF. After enrichment in buffered peptone water for 6 h, our developed qRT-PCR assay could reliably detect C. sakazakii when the inoculation level was as low as 2 CFU/25 g (0.08 CFU/g) in PIF. The growth of C. sakazakii could be inhibited by the presence of Lactobacillus pentosus and Bacillus cereus, which used a longer enrichment period before the isolation was accomplished. However, at 5 and 50 CFU/25 g inoculation levels of C. sakazakii in the presence of 4 × 10(6) CFU L. pentosus/25 g or of 2 × 10(4) CFU B. cereus/25 g, the qRT-PCR assay could detect the presence of Cronobacter even though these artificially spiked samples were negative in culture. Therefore, our results indicated that the qRT-PCR assay could detect samples containing inhibitors and could avoid false negatives by using an internal amplification control.

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

PubMed

Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

2016-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PubMed Central

Wang, Jiann-Hsiung; Chou, Shih-Jen; Li, Tsung-Hsien; Leu, Ming-Yih; Ho, Hsiao-Kuan

2017-01-01

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset. PMID:28970970

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas).

PubMed

Tsai, Ming-An; Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Li, Tsung-Hsien; Leu, Ming-Yih; Ho, Hsiao-Kuan; Yang, Wei Cheng

2017-01-01

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales ( Delphinapterus leucas ) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNA later ™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Characterization of miRNAs in response to short-term waterlogging in three inbred lines of Zea mays

USDA-ARS?s Scientific Manuscript database

To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and ...

Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani

USDA-ARS?s Scientific Manuscript database

Gene expression analysis requires the use of reference genes in the target species. The long yellow daylily is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asia. However, reference genes for use in RT-qPCR in this ...

Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

PubMed

Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

2016-09-01

There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A candidate gene for autoimmune myasthenia gravis

PubMed Central

Landouré, Guida; Knight, Melanie A.; Stanescu, Horia; Taye, Addis A.; Shi, Yijun; Diallo, Oumarou; Johnson, Janel O.; Hernandez, Dena; Traynor, Bryan J.; Biesecker, Leslie G.; Elkahloun, Abdel; Rinaldi, Carlo; Vincent, Angela; Willcox, Nick; Kleta, Robert; Fischbeck, Kenneth H.

2012-01-01

Objective: We sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis. Methods: We performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (RT-PCR) were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles in a heterozygote. Results: A region of shared homozygosity at chromosome 13q13.3–13q14.11 was found in 4 affected siblings and 1 unaffected sibling. A homozygous single nucleotide variant was found in the 3′-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%–60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability. Conclusion: These results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients. PMID:22744667

[Roles of KLF5 in inhibition TNFα-induced SK-BR-3 breast cancer cell apoptosis].

PubMed

Shi, Jianhong; Liu, Caiyun; Zhang, Anyi; Cui, Naipeng; Wang, Bing; Chen, Baoping; Ma, Zhenfeng

2014-07-08

To explore the expression levels and roles of Krüpple-like factor 5 (KLF5) in tumor necrosis factor α (TNFα)-induced SK-BR-3 breast cancer cells. SK-BR-3 breast cancer cells were stimulated by TNFα at different concentrations (0, 1, 5, 10, 20 µg/L) for specified durations (0, 6, 12, 24, 36 h). Western blot was performed to detect KLF5 protein levels. Then Western blot and quantitative real-time PCR (qRT-PCR) were used to detect the expression levels of apoptosis genes. Flow cytometry and qRT-PCR were used to observe the effects of exogenous KLF5 on TNFα-induced apoptosis of SK-BR-3 breast cancer cell. KLF5 expression levels significantly decreased in TNFα-stimulated SK-BR-3 breast cancer cells in a concentration- and time-dependent manner. Quantitative RT-PCR results showed that TNFα up-regulate apoptosis gene caspase 3, caspase 9 and bax expression levels and down-regulate bcl-1 level in SK-BR-3 cells. Adenovirus expression vectors of pAd-GFP and pAd-GFP-KLF5 were constructed and used to infect SK-BR-3 breast cancer cells. Over-expression of GFP-KLF5 inhibited apoptosis in TNFα-stimulated SK-BR-3 breast cancer cells. TNFα reduces KLF5 expression in SK-BR-3 breast cancer cells and KLF5 participates in TNFα-induced SK-BR-3 cell apoptosis.

Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines.

PubMed

Zhou, Hao; Chen, Shun; Qi, Yulin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

2015-10-01

Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1β, IL6, and IL18) and antiviral cytokines (IFNα, IFNγ) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFNα, IFNγ, IL1β, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFNα (P < 0.01), IFNγ (P < 0.01), IL1β (P < 0.01), IL6 (P < 0.01), and IL18 (P < 0.05). The established methods are important for scientific research and clinical applications, which require rapid and accurate results in a short period of time. The technique can potentially be used in the further research of goose molecular immunology, which will help us understand the interactions between hosts and pathogens. © 2015 Poultry Science Association Inc.

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions

PubMed Central

Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Sethuraman, Veeran; Yi, Xin; Zhong, Guohua

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin. PMID:29695976

Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Larvae of Spodoptera litura Exposed to Azadirachtin Stress Conditions.

PubMed

Shu, Benshui; Zhang, Jingjing; Cui, Gaofeng; Sun, Ranran; Sethuraman, Veeran; Yi, Xin; Zhong, Guohua

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura . In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

PubMed

Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

2016-07-15

Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.

Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity.

PubMed

Santos, Fabiane Igansi de Castro Dos; Marini, Naciele; Santos, Railson Schreinert Dos; Hoffman, Bianca Silva Fernandes; Alves-Ferreira, Marcio; de Oliveira, Antonio Costa

2018-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is a technique for gene expression profiling with high sensibility and reproducibility. However, to obtain accurate results, it depends on data normalization by using endogenous reference genes whose expression is constitutive or invariable. Although the technique is widely used in plant stress analyzes, the stability of reference genes for iron toxicity in rice (Oryza sativa L.) has not been thoroughly investigated. Here, we tested a set of candidate reference genes for use in rice under this stressful condition. The test was performed using four distinct methods: NormFinder, BestKeeper, geNorm and the comparative ΔCt. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. Valid reference genes were found for shoot (P2, OsGAPDH and OsNABP), root (OsEF-1a, P8 and OsGAPDH) and root+shoot (OsNABP, OsGAPDH and P8) enabling us to perform further reliable studies for iron toxicity in both indica and japonica subspecies. The importance of the study of other than the traditional endogenous genes for use as normalizers is also shown here.

Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity

PubMed Central

dos Santos, Fabiane Igansi de Castro; Marini, Naciele; dos Santos, Railson Schreinert; Hoffman, Bianca Silva Fernandes; Alves-Ferreira, Marcio

2018-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is a technique for gene expression profiling with high sensibility and reproducibility. However, to obtain accurate results, it depends on data normalization by using endogenous reference genes whose expression is constitutive or invariable. Although the technique is widely used in plant stress analyzes, the stability of reference genes for iron toxicity in rice (Oryza sativa L.) has not been thoroughly investigated. Here, we tested a set of candidate reference genes for use in rice under this stressful condition. The test was performed using four distinct methods: NormFinder, BestKeeper, geNorm and the comparative ΔCt. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. Valid reference genes were found for shoot (P2, OsGAPDH and OsNABP), root (OsEF-1a, P8 and OsGAPDH) and root+shoot (OsNABP, OsGAPDH and P8) enabling us to perform further reliable studies for iron toxicity in both indica and japonica subspecies. The importance of the study of other than the traditional endogenous genes for use as normalizers is also shown here. PMID:29494624

[Detection of Plasmodium falciparum by using magnetic nanoparticles separation-based quantitative real-time PCR assay].

PubMed

Wang, Fei; Tian, Yin; Yang, Jing; Sun, Fu-Jun; Sun, Ning; Liu, Bi-Yong; Tian, Rui; Ge, Guang-Lu; Zou, Ming-qiang; Deng, Cong-liang; Liu, Yi

2014-10-01

To establish a magnetic nanoparticles separation-based quantitative real-time PCR (RT-PCR) assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. According to the conserved sequences of the P. falciparum genome 18SrRNA, the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard, fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. The relationship between the threshold cycle (Ct) and logarithm of initial templates copies was linear over a range of 2.5 x 10(1) to 2.5 x 10(8) copies/μl (R2 = 0.999). Among 13 subjects of entry frontier, a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations (microscopic examinations and rapid tests). This assay shows a high sensitivity in detection of P. falciparum, with rapid and accurate characteristics, and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus.

PubMed

Shen, Xin-Xin; Qiu, Fang-Zhou; Zhao, Huai-Long; Yang, Meng-Jie; Hong, Liu; Xu, Song-Tao; Zhou, Shuai-Feng; Li, Gui-Xia; Feng, Zhi-Shan; Ma, Xue-Jun

2018-03-01

The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10 -8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.

PubMed

Kurakawa, Takashi; Ogata, Kiyohito; Tsuji, Hirokazu; Kado, Yukiko; Takahashi, Takuya; Kida, Yumi; Ito, Masahiro; Okada, Nobuhiko; Nomoto, Koji

2015-04-01

Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel small RNA S042 increases acid tolerance in Lactococcus lactis F44.

PubMed

Wu, Hao; Song, Shunyi; Tian, Kairen; Zhou, Dandan; Wang, Binbin; Liu, Jiaheng; Zhu, Hongji; Qiao, Jianjun

2018-06-07

Lactococcus lactis, a gram-positive bacterium, encounters various environmental stresses, especially acid stress, during fermentation. Small RNAs (sRNAs) that serve as regulators at post-transcriptional level play important roles in acid stress response. Here, a novel sRNA S042 was identified by RNA-Seq, RT-PCR and Northern blot. The transcription level of s042 was upregulated 2.29-fold under acid stress by Quantitative RT-PCR (qRT-PCR) analysis. Acid tolerance assay showed that overexpressing s042 increased the survival rate of L. lactis F44 and deleting s042 significantly inhibited the viability under acidic conditions. Moreover, the targets were predicted by online software and four genes were chosen as candidates. Among them, argR (arginine regulator) and accD (acetyl-CoA carboxylase carboxyl transferase subunit beta) were validated to be the direct targets activated by S042 through reporter fusion assay. The regulatory mechanism between S042 and its targets was further investigated through Bioinformatics and qRT-PCR. This study served to highlight the role of the novel sRNA S042 in acid resistance of L. lactis and provided new insights into the response mechanism of acid stress. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

PubMed

Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

2009-05-01

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.

PubMed

Paten, A M; Pain, S J; Peterson, S W; Blair, H T; Kenyon, P R; Dearden, P K; Duncan, E J

2014-08-01

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland. Copyright © 2014 the American Physiological Society.

Aberrant expression of long noncoding RNAs in cumulus cells isolated from PCOS patients.

PubMed

Huang, Xin; Hao, Cuifang; Bao, Hongchu; Wang, Meimei; Dai, Huangguan

2016-01-01

To describe the long noncoding RNA (lncRNA) profiles in cumulus cells isolated from polycystic ovary syndrome (PCOS) patients by employing a microarray and in-depth bioinformatics analysis. This information will help us understand the occurrence and development of PCOS. In this study, we used a microarray to describe lncRNA profiles in cumulus cells isolated from ten patients (five PCOS and five normal women). Several differentially expressed lncRNAs were chosen to validate the microarray results by quantitative RT-PCR (qRT-PCR). Then, the differentially expressed lncRNAs were classified into three subgroups (HOX loci lncRNA, enhancer-like lncRNA, and lincRNA) to deduce their potential features. Furthermore, a lncRNA/mRNA co-expression network was constructed by using the Cytoscape software (V2.8.3, http://www.cytoscape.org/ ). We observed that 623 lncRNAs and 260 messenger RNAs (mRNAs) were significantly up- or down-regulated (≥2-fold change), and these differences could be used to discriminate cumulus cells of PCOS from those of normal patients. Five differentially expressed lncRNAs (XLOC_011402, ENST00000454271, ENST00000433673, ENST00000450294, and ENST00000432431) were selected to validate the microarray results using quantitative RT-PCR (qRT-PCR). The qRT-PCR results were consistent with the microarray data. Further analysis indicated that many differentially expressed lncRNAs were transcribed from chromosome 2 and may act as enhancers to regulate their neighboring protein-coding genes. Forty-three lncRNAs and 29 mRNAs were used to construct the coding-non-coding gene co-expression network. Most pairs positively correlated, and one mRNA correlated with one or more lncRNAs. Our study is the first to determine genome-wide lncRNA expression patterns in cumulus cells isolated from PCOS patients by microarray. The results show that clusters of lncRNAs were aberrantly expressed in cumulus cells of PCOS patients compared with those of normal women, which revealed that lncRNAs differentially expressed in PCOS and normal women may contribute to the occurrence of PCOS and affect oocyte development.

Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: A targeted rtPCR array approach for defining relative potency

EPA Science Inventory

Male rat fetuses exposed to certain phthalate esters (PEs) during sexual differentiation display reproductive tract malformations due to reductions in testosterone (T) production and the expression of steroidogenesis-and INSL3-related genes. In the current study} we used a 96well...

Evaluation of genomic biomarkers and relative potency of phthalate-induced male reproductive developmental toxicity using a targeted rtPCR array approach**

EPA Science Inventory

Exposure to certain phthalate esters (PEs) during sexual differentiation induces reproductive tract malformations in male rats due to reductions in fetal testicular testosterone (T) production and expression of steroidogenesis-and insl3-related genes. In the current study, we use...

Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

PubMed

Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

2015-07-01

Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

Facing the problem of "false positives": re-assessment and improvement of a multiplex RT-PCR procedure for the diagnosis of A. flavus mycotoxin producers.

PubMed

Degola, F; Berni, E; Spotti, E; Ferrero, I; Restivo, F M

2009-02-28

The aim of our research project was to consolidate a multiplex RT-PCR protocol to detect aflatoxigenic strains of Aspergillus flavus. Several independent A. flavus strains were isolated from corn and flour samples from the North of Italy and from three European countries. Aflatoxin producing/not producing phenotype was assessed by qualitative and quantitative assays at day five of growth in aflatoxin inducing conditions. Expression of 16 genes belonging to the aflatoxin cluster was assayed by multiplex or monomeric RT-PCR. There is a good correlation between gene expression and aflatoxin production. Strains that apparently transcribed all the relevant genes but did not release aflatoxin in the medium ("false positives") were re-assessed for mycotoxin production after extended growth in inducing condition. All the "false positive" strains in actual fact were positive when aflatoxin determination was performed after 10 days of growth. These strains should then be re-classified as "slow aflatoxin accumulators". To optimise the diagnostic procedure, a quintuplex RT-PCR procedure was designed consisting of a primer set directed against four informative aflatoxin cluster genes and the beta-tubulin gene as an internal amplification control. In conclusion we have provided evidence for the robustness and reliability of our RT-PCR protocol in discriminating mycotoxin producer from non-producer strains of A. flavus. and the molecular procedure we devised is a promising tool with which to screen and control the endemic population of A. flavus colonising different areas of the World.

Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF)

PubMed Central

Quilter, C.R.; Karcanias, A.C.; Bagga, M.R.; Duncan, S.; Murray, A.; Conway, G.S.; Sargent, C.A.; Affara, N.A.

2013-01-01

BACKGROUND Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization. METHODS In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)–PCR. RESULTS A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes. CONCLUSIONS The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF. PMID:20570974

Relationship Between Ebola Virus Real-Time Quantitative Polymerase Chain Reaction-Based Threshold Cycle Value and Virus Isolation From Human Plasma.

PubMed

Spengler, Jessica R; McElroy, Anita K; Harmon, Jessica R; Ströher, Ute; Nichol, Stuart T; Spiropoulou, Christina F

2015-10-01

We performed a longitudinal analysis of plasma samples obtained from 4 patients with Ebola virus (EBOV) disease (EVD) to determine the relationship between the real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based threshold cycle (Ct) value and the presence of infectious EBOV. EBOV was not isolated from plasma samples with a Ct value of >35.5 or >12 days after onset of symptoms. EBOV was not isolated from plasma samples in which anti-EBOV nucleoprotein immunoglobulin G was detected. These data demonstrate the utility of interpreting qRT-PCR results in the context of the course of EBOV infection and associated serological responses for patient-management decisions. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Novel chromosomal rearrangements and break points at the t(6;9) in salivary adenoid cystic carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome.

PubMed

Mitani, Yoshitsugu; Rao, Pulivarthi H; Futreal, P Andrew; Roberts, Dianna B; Stephens, Philip J; Zhao, Yi-Jue; Zhang, Li; Mitani, Mutsumi; Weber, Randal S; Lippman, Scott M; Caulin, Carlos; El-Naggar, Adel K

2011-11-15

To investigate the molecular genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Multimolecular and genetic techniques complemented with massive pair-ended sequencing and single-nucleotide polymorphism array analyses were used on tumor specimens from 30 new and 52 previously analyzed fusion transcript-negative ACCs by reverse transcriptase PCR (RT-PCR). MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients' survival. The FISH analysis showed 34 of 82 (41.5%) fusion-positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% whereas fusion transcript forming incidence was 38.2%. Significant statistical association between the 5' MYB transcript expression and patient survival was found. We conclude that: (i) t(6;9) results in complex genetic and molecular alterations in ACC, (ii) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, (iii) noncanonical MYB-NFIB gene fusions occur in a subset of tumors, (iv) high MYB expression correlates with worse patient survival.

MLF1 interacting protein: a potential gene therapy target for human prostate cancer?

PubMed

Zhang, Lei; Ji, Guoqing; Shao, Yuzhang; Qiao, Shaoyi; Jing, Yuming; Qin, Rongliang; Sun, Huiming; Shao, Chen

2015-02-01

Here, we investigated the role of one gene that has been previously associated with human prostate carcinoma cells-myelodysplasia/myeloid leukemia factor 1 interacting protein (MLF1IP)-in order to better ascertain its role in human prostate carcinogenesis. The prostate cancer cell line PC-3 was lentivirally transfected to silence endogenous MLF1IP gene expression, which was confirmed by real-time quantitative PCR (RT-qPCR). Cellomics ArrayScan VTI imaging and MTT assays were conducted to assess cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry. Colony formation was assessed by fluorescence microscopy. MLF1IP gene expression was also analyzed by RT-qPCR in sixteen prostate cancer tissue samples and six healthy control prostate tissue samples from human patients. Cell proliferation was significantly inhibited in MLF1IP-silenced cells relative to control cells. G1 phase, S and G2/M phase cell counts were not significantly changed in MLF1IP-silenced cells relative to control cells. Apoptosis was significantly increased in MLF1IP-silenced cells, while MLF1IP-silenced cells displayed a significantly reduced number of cell colonies, compared to control cells. The 16 human prostate cancer tissue samples revealed no clear upregulation or downregulation in MLF1IP gene expression. MLF1IP significantly promotes prostate cancer cell proliferation and colony formation and significantly inhibits apoptosis without affecting cell cycle phase arrest. Further study is required to conclusively determine whether MLF1IP is upregulated in human prostate cancer tumors and to determine the precise cellular mechanism(s) for MLF1IP in prostate carcinogenesis.

IL-13 Stimulates Proliferation and Expression of Mucin and Immunomodulatory Genes in Cultured Conjunctival Goblet Cells.

PubMed

Tukler Henriksson, Johanna; Coursey, Terry G; Corry, David B; De Paiva, Cintia S; Pflugfelder, Stephen C

2015-07-01

To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes. Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 μL per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array. The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-β at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment. This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix(®) and RotaTeq(®) vaccine strains in stool samples.

PubMed

Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D

2014-01-01

Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix(®) and RotaTeq(®) are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix(®) and RotaTeq(®) vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix(®) and RotaTeq(®) vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix(®) (NSP2, VP4) and RotaTeq(®) (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix(®) NSP2 and VP4 qRT-PCR assays exhibited 92-100% sensitivity, 99-100% specificity, 94-105% efficiency, and a limit of detection of 2-3 copies per reaction. RotaTeq(®) VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94-100% specificity, 91-102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix(®) and RotaTeq(®) vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE.

Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix® and RotaTeq® vaccine strains in stool samples

PubMed Central

Gautam, Rashi; Esona, Mathew D; Mijatovic-Rustempasic, Slavica; Ian Tam, Ka; Gentsch, Jon R; Bowen, Michael D

2014-01-01

Group A rotaviruses (RVA) are the leading cause of severe diarrhea in young children worldwide. Two live-attenuated RVA vaccines, Rotarix® and RotaTeq® are recommended by World Health Organization (WHO) for routine immunization of all infants. Rotarix® and RotaTeq® vaccines have substantially reduced RVA associated mortality but occasionally have been associated with acute gastroenteritis (AGE) cases identified in vaccinees and their contacts. High-throughput assays are needed to monitor the prevalence of vaccine strains in AGE cases and emergence of new vaccine-derived strains following RVA vaccine introduction. In this study, we have developed quantitative real-time RT-PCR (qRT-PCR) assays for detection of Rotarix® and RotaTeq® vaccine components in stool samples. Real-time RT-PCR assays were designed for vaccine specific targets in the genomes of Rotarix® (NSP2, VP4) and RotaTeq® (VP6, VP3-WC3, VP3-human) and validated on sequence confirmed stool samples containing vaccine strains, wild-type RVA strains, and RVA-negative stools. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Rotarix® NSP2 and VP4 qRT-PCR assays exhibited 92–100% sensitivity, 99–100% specificity, 94–105% efficiency, and a limit of detection of 2–3 copies per reaction. RotaTeq® VP6, VP3-WC3, and VP3-human qRT-PCR assays displayed 100% sensitivity, 94–100% specificity, 91–102% efficiency and limits of detection of 1 copy, 2 copies, and 140 copies, respectively. These assays permit rapid identification of Rotarix® and RotaTeq® vaccine components in stool samples from clinical and surveillance studies and will be helpful in determining the frequency of vaccine strain-associated AGE. PMID:24342877

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

PubMed

Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

2012-01-01

Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

Bluetongue virus surveillance in the Islamic Republic of Mauritania: Is serotype 26 circulating among cattle and dromedaries?

PubMed

Lorusso, Alessio; Baba, Doumbia; Spedicato, Massimo; Teodori, Liana; Bonfini, Barbara; Marcacci, Maurilia; Di Provvido, Andrea; Isselmou, Katia; Marini, Valeria; Carmine, Irene; Scacchia, Massimo; Di Sabatino, Daria; Petrini, Antonio; Bezeid, Beyatt Ahmed; Savini, Giovanni

2016-06-01

In March 2013, EDTA-blood and serum samples were collected from 119 cattle and 159 dromedaries at the slaughterhouse of Nouakchott, the capital city of the Islamic Republic of Mauritania. Serum samples were screened for the presence of Bluetongue (BT) antibodies by competitive ELISA (cELISA). Positive samples were then tested by serum-neutralization (SN) to determine BTV serotype. RNA from blood samples was first tested by a genus-specific quantitative RT-PCR assay which is able to detect all 27 existing BTV serotypes (RT-qPCR1-27). Positive samples were further screened by a RT-qPCR assay which, instead, is able to detect the classical 24 BTV serotypes only (RT-qPCR1-24). Of the 278 serum samples tested, 177 (mean=63.7%; 95% CI: 57.9%-69.1%) resulted positive by cELISA. Of these, 69 were from cattle (mean=58.0%; 95% CI: 49.0%-66.5%) and 108 from dromedaries (mean=67.9%; 95% CI: 60.3%-74.7%). BTV-26 neutralizing antibodies were by far the most frequently found as they were detected in 146 animals with titres ranging from 1:10 to 1:80. Out of 278 blood samples, 25 (mean=9.0%; 95% CI: 6.2%-12.9%) were found positive for BTV by RT-qPCR1-27, 20 (mean=16.8%; 95% CI: 11.2%-24.6%) were from cattle and 5 (mean=3.1%; 95% CI: 1.4%-7.1%) from dromedaries. When tested by RT-qPCR1-24 the 25 BTV positive samples were negative. Unfortunately, no genetic information by molecular typing or by next generation sequencing has been obtained as for the very low levels of RNA in the blood samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and Evaluation of Reliable Reference Genes in the Medicinal Fungus Shiraia bambusicola.

PubMed

Song, Liang; Li, Tong; Fan, Li; Shen, Xiao-Ye; Hou, Cheng-Lin

2016-04-01

The stability of reference genes plays a vital role in real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, which is generally regarded as a convenient and sensitive tool for the analysis of gene expression. A well-known medicinal fungus, Shiraia bambusicola, has great potential in the pharmaceutical, agricultural and food industries, but its suitable reference genes have not yet been determined. In the present study, 11 candidate reference genes in S. bambusicola were first evaluated and validated comprehensively. To identify the suitable reference genes for qRT-PCR analysis, three software-based algorithms, geNorm, NormFinder and Best Keeper, were applied to rank the tested genes. RNA samples were collected from seven fermentation stages using different media (potato dextrose or Czapek medium) and under different light conditions (12-h light/12-h dark and all-dark). The three most appropriate reference genes, ubi, tfc and ags, were able to normalize the qRT-PCR results under the culturing conditions of 12-h light/12-h dark, whereas the other three genes, vac, gke and acyl, performed better in the culturing conditions of all-dark growth. Therefore, under different light conditions, at least two reference genes (ubi and vac) could be employed to assure the reliability of qRT-PCR results. For both the natural culture medium (the most appropriate genes of this group: ubi, tfc and ags) and the chemically defined synthetic medium (the most stable genes of this group: tfc, vac and ef), the tfc gene remained the best gene used for normalizing the gene expression found with qRT-PCR. It is anticipated that these results would improve the selection of suitable reference genes for qRT-PCR assays and lay the foundation for an accurate analysis of gene expression in S. bambusicola.

Identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells.

PubMed

Johnston, Daniel S; Jelinsky, Scott A; Zhi, Yu; Finger, Joshua N; Kopf, Gregory S; Wright, William W

2007-12-01

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Curcumin may serve an anticancer role in human osteosarcoma cell line U-2 OS by targeting ITPR1.

PubMed

Luo, Zhanpeng; Li, Dawei; Luo, Xiaobo; Li, Litao; Gu, Suxi; Yu, Long; Ma, Yuanzheng

2018-04-01

The present study aimed to determine the mechanisms of action of curcumin in osteosarcoma. Human osteosarcoma U-2 OS cells was purchased from the Cell Bank of the Chinese Academy of Sciences. RNA sequencing analysis was performed for 2 curcumin-treated samples and 2 control samples using Illumina deep sequencing technology. The differentially expressed genes were identified using Cufflink software. Enrichment and protein-protein interaction network analyses were performed separately using cluster Profiler package and Cytoscape software to identify key genes. Then, the mRNA levels of key genes were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in U-2 OS cells. Finally, cell apoptosis, proliferation, migration and invasion arrays were performed. In total, 201 DEGs were identified in the curcumin-treated group. EEF1A1 (degree=88), ATF7IP, HIF1A, SMAD7, CLTC, MCM10, ITPR1, ADAM15, WWP2 and ATP5C1, which were enriched in 'biological process', exhibited higher degrees than other genes in the PPI network. RT-qPCR demonstrated that treatment with curcumin was able to significantly increase the levels of CLTC and ITPR1 mRNA in curcumin-treated cells compared with control. In addition, targeting ITPR1 with curcumin significantly promoted apoptosis and suppressed proliferation, migration and invasion. Targeting ITPR1 via curcumin may serve an anticancer role by mediating apoptosis, proliferation, migration and invasion in U-2 OS cells.

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

2015-01-01

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

PubMed

Kupsch, C; Ohst, T; Pankewitz, F; Nenoff, P; Uhrlaß, S; Winter, I; Gräser, Y

2016-08-01

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection

PubMed Central

Zaas, Aimee K.; Burke, Thomas; Chen, Minhua; McClain, Micah; Nicholson, Bradly; Veldman, Timothy; Tsalik, Ephraim L.; Fowler, Vance; Rivers, Emanuel P.; Otero, Ronny; Kingsmore, Stephen F.; Voora, Deepak; Lucas, Joseph; Hero, Alfred O.; Carin, Lawrence; Woods, Christopher W.; Ginsburg, Geoffrey S.

2014-01-01

Improved ways to diagnose acute respiratory viral infections could decrease inappropriate antibacterial use and serve as a vital triage mechanism in the event of a potential viral pandemic. Measurement of the host response to infection is an alternative to pathogen-based diagnostic testing and may improve diagnostic accuracy. We have developed a host-based assay with a reverse transcription polymerase chain reaction (RT-PCR) TaqMan low-density array (TLDA) platform for classifying respiratory viral infection. We developed the assay using two cohorts experimentally infected with influenza A H3N2/Wisconsin or influenza A H1N1/Brisbane, and validated the assay in a sample of adults presenting to the emergency department with fever (n = 102) and in healthy volunteers (n = 41). Peripheral blood RNA samples were obtained from individuals who underwent experimental viral challenge or who presented to the emergency department and had microbiologically proven viral respiratory infection or systemic bacterial infection. The selected gene set on the RT-PCR TLDA assay classified participants with experimentally induced influenza H3N2 and H1N1 infection with 100 and 87% accuracy, respectively. We validated this host gene expression signature in a cohort of 102 individuals arriving at the emergency department. The sensitivity of the RT-PCR test was 89% [95% confidence interval (CI), 72 to 98%], and the specificity was 94% (95% CI, 86 to 99%). These results show that RT-PCR–based detection of a host gene expression signature can classify individuals with respiratory viral infection and sets the stage for prospective evaluation of this diagnostic approach in a clinical setting. PMID:24048524

Quantitative estimation of Nipah virus replication kinetics in vitro

PubMed Central

Chang, Li-Yen; Ali, AR Mohd; Hassan, Sharifah Syed; AbuBakar, Sazaly

2006-01-01

Background Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR® Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay. Results The qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious. Conclusion The SYBR® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection. PMID:16784519

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells

Treesearch

Andrew F. Page; Rakesh Minocha; Subhash C. Minocha

2012-01-01

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied...

Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

PubMed

Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

2016-08-01

Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect the two viruses and differentiate PEDV from PDCoV. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of EML4-ALK fusion gene in Chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique.

PubMed

Fu, Sha; Wang, Fang; Shao, Qiong; Zhang, Xu; Duan, Li-Ping; Zhang, Xiao; Zhang, Li; Shao, Jian-Yong

2015-04-01

Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

PubMed

Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

2016-04-07

Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae).

PubMed

Zhang, Songdou; An, Shiheng; Li, Zhen; Wu, Fengming; Yang, Qingpo; Liu, Yichen; Cao, Jinjun; Zhang, Huaijiang; Zhang, Qingwen; Liu, Xiaoxia

2015-01-25

Recent studies have focused on determining functional genes and microRNAs in the pest Helicoverpa armigera (Lepidoptera: Noctuidae). Most of these studies used quantitative real-time PCR (qRT-PCR). Suitable reference genes are necessary to normalize gene expression data of qRT-PCR. However, a comprehensive study on the reference genes in H. armigera remains lacking. Twelve candidate reference genes of H. armigera were selected and evaluated for their expression stability under different biotic and abiotic conditions. The comprehensive stability ranking of candidate reference genes was recommended by RefFinder and the optimal number of reference genes was calculated by geNorm. Two target genes, thioredoxin (TRX) and Cu/Zn superoxide dismutase (SOD), were used to validate the selection of reference genes. Results showed that the most suitable candidate combinations of reference genes were as follows: 28S and RPS15 for developmental stages; RPS15 and RPL13 for larvae tissues; EF and RPL27 for adult tissues; GAPDH, RPL27, and β-TUB for nuclear polyhedrosis virus infection; RPS15 and RPL32 for insecticide treatment; RPS15 and RPL27 for temperature treatment; and RPL32, RPS15, and RPL27 for all samples. This study not only establishes an accurate method for normalizing qRT-PCR data in H. armigera but also serve as a reference for further study on gene transcription in H. armigera and other insects. Copyright © 2014 Elsevier B.V. All rights reserved.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

PubMed Central

Hashemipetroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

2018-01-01

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species PMID:29443017

Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water.

PubMed

Randazzo, W; López-Gálvez, Francisco; Allende, A; Aznar, R; Sánchez, G

2016-07-16

Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoculated lettuce. Pretreatment with 50μM PMAxx and 0.5% Triton X-100 (Triton) for 10min reduced the signal of thermally inactivated NoV by ca. 1.8 logs for both genogroups in lettuce concentrates. Additionally, this pretreatment reduced the signal of thermally inactivated NoV GI between 1.4 and 1.9 logs in spinach and romaine and lamb's lettuces and by >2 logs for NoV GII in romaine and lamb's lettuce samples. Moreover this pretreatment was satisfactorily applied to naturally-contaminated water samples with NoV GI and GII. Based on the obtained results this pretreatment has the potential to be integrated in routine diagnoses to improve the interpretation of positive NoV results obtained by RT-qPCR. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of EVI1 transcript levels in acute myeloid leukemia by RT-qPCR analysis: A study by the ALFA Group.

PubMed

Smol, Thomas; Nibourel, Olivier; Marceau-Renaut, Alice; Celli-Lebras, Karine; Berthon, Céline; Quesnel, Bruno; Boissel, Nicolas; Terré, Christine; Thomas, Xavier; Castaigne, Sylvie; Dombret, Hervé; Preudhomme, Claude; Renneville, Aline

2015-12-01

EVI1 overexpression confers poor prognosis in acute myeloid leukemia (AML). Quantification of EVI1 expression has been mainly assessed by real-time quantitative PCR (RT-qPCR) based on relative quantification of EVI1-1D splice variant. In this study, we developed a RT-qPCR assay to perform quantification of EVI1 expression covering the different splice variants. A sequence localized in EVI1 exons 14 and 15 was cloned into plasmids that were used to establish RT-qPCR standard curves. Threshold values to define EVI1 overexpression were determined using 17 bone marrow (BM) and 31 peripheral blood (PB) control samples and were set at 1% in BM and 0.5% in PB. Samples from 64 AML patients overexpressing EVI1 included in the ALFA-0701 or -0702 trials were collected at diagnosis and during follow-up (n=152). Median EVI1 expression at AML diagnosis was 23.3% in BM and 3.6% in PB. EVI1 expression levels significantly decreased between diagnostic and post-induction samples, with an average variation from 21.6% to 3.56% in BM and from 4.0% to 0.22% in PB, but did not exceed 1 log10 reduction. Our study demonstrates that the magnitude of reduction in EVI1 expression levels between AML diagnosis and follow-up is not sufficient to allow sensitive detection of minimal residual disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Digital PCR provides absolute quantitation of viral load for an occult RNA virus.

PubMed

White, Richard Allen; Quake, Stephen R; Curr, Kenneth

2012-01-01

Using a multiplexed LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a prefabricated microfluidic device that monitored absolute viral load in native and immortalized cell lines, overall precision of detection, and the absolute detection limit of an occult RNA virus GB Virus Type C (GBV-C). RT-dPCR had on average a 10% lower overall coefficient of variation (CV, a measurement of precision) for viral load testing than RT-qPCR and had a higher overall detection limit, able to quantify as low as three 5'-UTR molecules of GBV-C genome. Two commercial high-yield in vitro transcription kits (T7 Ribomax Express by Promega and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA genome. THP-1 cells (a model of monocytic derived cells) were transfected with GBV-C, yielding infectious virions that replicated over a 120h time course and could be infected directly. This study provides the first evidence of GBV-C replication in monocytic derived clonal cells. Thus far, it is the only study using a microfluidic device that measures directly viral load of mammalian RNA virus in a digital format without need for a standard curve. Copyright © 2011 Elsevier B.V. All rights reserved.

Assessment of phthalate-induced changes in fetal rat testis gene expression using an rt-PCR array

EPA Science Inventory

Lambright, CS’, Sampson, H2, Furr, i1, Evans, N’, Hannas, B’, Gray, LE, Jr.’, VS Wilson1. 1USEPA, NHEERL, RTP, NC, 2USEPA, NHEERL, RTP, NC, ORISE Fellow. Phthalate esters (PE) such as diethyl hexyl phthalate (DEHP) produce reproductive malformations in male rodents by reduction o...

Prognostic role of a multigene reverse transcriptase-PCR assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy.

PubMed

Esteva, Francisco J; Sahin, Aysegul A; Cristofanilli, Massimo; Coombes, Kevin; Lee, Sang-Joon; Baker, Joffre; Cronin, Maureen; Walker, Michael; Watson, Drew; Shak, Steven; Hortobagyi, Gabriel N

2005-05-01

To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene expression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tissue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. We obtained archival paraffin-embedded tissue from patients with invasive breast cancer but no axillary lymph node involvement who had received no adjuvant systemic therapy and been followed for at least 5 years. RNA was extracted from three 10-microm-thick sections. The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the ability of the test to accurately predict distant recurrence-free survival in this population. We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21 gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status was noted. RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to develop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy.

A Comparative Proteomic Analysis of the Buds and the Young Expanding Leaves of the Tea Plant (Camellia sinensis L.)

PubMed Central

Li, Qin; Li, Juan; Liu, Shuoqian; Huang, Jianan; Lin, Haiyan; Wang, Kunbo; Cheng, Xiaomei; Liu, Zhonghua

2015-01-01

Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants. PMID:26096006

Improving Ethanol Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

PubMed Central

Yeow, Jianwei; Wang, Ivy; Zhang, Hongfang; Song, Hao; Jiang, Rongrong

2013-01-01

A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1– E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h−1 in 62 g/L ethanol, higher than that of the control at 0.06 h−1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol. PMID:23469036

Transcript patterns of Phanerochaete chrysosporium genes in organopollutant contaminated soils and in wood

Treesearch

Amber Vanden Wymelenberg; Bernard Janse; Jill Gaskell; Diane Dietrich; Marcelo Vallim; Dan Cullen

1998-01-01

We describe here recent methods for quantitative assessment of specific P. chrysosporium mRNAs in organopollutant contaminated soils and in Aspen wood chips. Magnetic capture techniques were used to rapidly purify poly(A)-RNA, and quantitative RT-PCR protocols were developed for all known lignin peroxidase (lip) and cellobiohydrolase (cbh1) genes. The methodology is...

Evaluation of the stability of reference genes in bone mesenchymal stem cells from patients with avascular necrosis of the femoral head.

PubMed

Wang, X N; Yang, Q W; Du, Z W; Yu, T; Qin, Y G; Song, Y; Xu, M; Wang, J C

2016-05-25

This study aimed to evaluate 12 genes (18S, GAPDH, B2M, ACTB, ALAS1, GUSB, HPRT1, PBGD, PPIA, PUM1, RPL29, and TBP) for their reliability and stability as reference sequences for real-time quantitative PCR (RT-qPCR) in bone marrow-derived mesenchymal stem cells (BMSCs) isolated from patients with avascular necrosis of the femoral head (ANFH). BMSCs were isolated from 20 ANFH patients divided into four groups according to etiology, and four donors with femoral neck fractures. Total RNA was isolated from BMSCs and reverse transcribed into complementary DNA, which served as a template for RT-qPCR. Three commonly used programs were then used to analyze the results. Reference gene expression varied within each group, between specific groups, and among all five groups. Based on comparisons of all five groups, two of the programs used suggested that HPRT1 was the most stable reference gene, while 18S and ACTB were the most variable. Among the 12 candidate reference genes, HPRT1 exhibited the greatest reliability, followed by PPIA. Thus, these sequences could be used as references for the normalization of RT-qPCR results.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

PubMed

Gao, Gang; Xu, Xiao-Xia; Yu, Jing; Li, Lin-Miao; Ju, Wen-Yan; Jin, Feng-Liang; Freed, Shoaib

2016-09-01

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella. © 2016 Wiley Periodicals, Inc.

Antimicrobial peptide gene expression in periodontitis patients: A pilot study.

PubMed

Jourdain, Marie-Laure; Pierrard, Loïc; Kanagaratnam, Lukshe; Velard, Frédéric; Sergheraert, Johan; Lefèvre, Benoît; Gangloff, Sophie C; Braux, Julien

2018-05-01

Antimicrobial peptides (AMPs) are one of the most active components of innate immunity and have characteristics that could place them at the heart of the pathogenesis of periodontal disease. This study investigated differences in the expression of AMP coding genes obtained using a simple harvesting technique, gingival smear, between two groups of patients: chronic periodontitis subjects versus healthy ones. Twenty-three patients were enrolled in two groups: 12 were diagnosed with moderate or severe generalized chronic periodontitis, and 11 were diagnosed as clinically healthy. Gingival smears were retrieved and studied using reverse transcription-quantitative PCR (RT-qPCR) after mRNA purification. Fifteen gene expressions were obtained using real-time RT-qPCR. Three AMP genes, histatin 3 (HTN3), α-defensin 4 (DEFA4) and lysozyme C (LYZ), presented different expression levels in periodontitis patients compared with healthy subjects. The relative expression level of DEFA4 appeared to be a protective factor against periodontitis. Gingival smears studied by RT-qPCR may be used to assess the expression of AMPs coding genes. A lack of expression of DEFA4 could be a potential indicator of periodontitis status. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.

PubMed

Gloghini, Annunziata; Canal, Barbara; Klein, Ulf; Dal Maso, Luigino; Perin, Tiziana; Dalla-Favera, Riccardo; Carbone, Antonino

2004-11-01

In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.

Utilizing Low-Volume Aqueous Acoustic Transfer with the Echo 525 to Enable Miniaturization of qRT-PCR Assay.

PubMed

Agrawal, Sony; Cifelli, Steven; Johnstone, Richard; Pechter, David; Barbey, Deborah A; Lin, Karen; Allison, Tim; Agrawal, Shree; Rivera-Gines, Aida; Milligan, James A; Schneeweis, Jonathan; Houle, Kevin; Struck, Alice J; Visconti, Richard; Sills, Matthew; Wildey, Mary Jo

2016-02-01

Quantitative reverse transcription PCR (qRT-PCR) is a valuable tool for characterizing the effects of inhibitors on viral replication. The amplification of target viral genes through the use of specifically designed fluorescent probes and primers provides a reliable method for quantifying RNA. Due to reagent costs, use of these assays for compound evaluation is limited. Until recently, the inability to accurately dispense low volumes of qRT-PCR assay reagents precluded the routine use of this PCR assay for compound evaluation in drug discovery. Acoustic dispensing has become an integral part of drug discovery during the past decade; however, acoustic transfer of microliter volumes of aqueous reagents was time consuming. The Labcyte Echo 525 liquid handler was designed to enable rapid aqueous transfers. We compared the accuracy and precision of a qPCR assay using the Labcyte Echo 525 to those of the BioMek FX, a traditional liquid handler, with the goal of reducing the volume and cost of the assay. The data show that the Echo 525 provides higher accuracy and precision compared to the current process using a traditional liquid handler. Comparable data for assay volumes from 500 nL to 12 µL allowed the miniaturization of the assay, resulting in significant cost savings of drug discovery and process streamlining. © 2015 Society for Laboratory Automation and Screening.

Internalization of Murine Norovirus 1 by Lactuca sativa during Irrigation ▿

PubMed Central

Wei, Jie; Jin, Yan; Sims, Tom; Kniel, Kalmia E.

2011-01-01

Romaine lettuce (Lactuca sativa) was grown hydroponically or in soil and challenged with murine norovirus 1 (MNV) under two conditions: one mimicking a severe one-time contamination event and another mimicking a lower level of contamination occurring over time. In each condition, lettuce was challenged with MNV delivered at the roots. In the first case, contamination occurred on day one with 5 × 108 reverse transcriptase quantitative PCR (RT-qPCR) U/ml MNV in nutrient buffer, and irrigation water was replaced with virus-free buffer every day for another 4 days. In the second case, contamination with 5 × 105 RT-qPCR U/ml MNV (freshly prepared) occurred every day for 5 days. Virus had a tendency to adsorb to soil particles, with a small portion suspended in nutrient buffer; e.g., ∼8 log RT-qPCR U/g MNV was detected in soil during 5 days of challenge with virus inoculums of 5 × 108 RT-qPCR U/ml at day one, but <6 log was found in nutrient buffer on days 3 and 5. For hydroponically grown lettuce, ∼3.4 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in some lettuce leaf samples after 5 days at high MNV inoculums, significantly higher than the internalized virus concentration (∼2.6 log) at low inoculums (P < 0.05). For lettuce grown in soil, approximately 2 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in lettuce with both high and low inoculums, showing no significant difference. For viral infectivity, infectious MNV was found in lettuce samples challenged with high virus inoculums grown hydroponically and in soil but not in lettuce grown with low virus inoculums. Lettuce grown hydroponically was further incubated in 99% and 70% relative humidities (RH) to evaluate plant transpiration relative to virus uptake. More lettuce samples were found positive for MNV at a significantly higher transpiration rate at 70% RH, indicating that transpiration might play an important role in virus internalization into L. sativa. PMID:21296944

Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity.

PubMed

Li, B; Matter, E K; Hoppert, H T; Grayson, B E; Seeley, R J; Sandoval, D A

2014-02-01

Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies.

Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don.

PubMed

Xiao, Zheng; Sun, Xiaobo; Liu, Xiaoqing; Li, Chang; He, Lisi; Chen, Shangping; Su, Jiale

2016-01-01

The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1- α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin ( TUB ) was the least stable. ACT5 (actin), RPL3 , 18S , and EF1- α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle . Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1- α, 18S , ACT5 , RPL3 , and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle .

Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR.

PubMed

Schmalz, Gerhard; Tsigaras, Sandra; Rinke, Sven; Kottmann, Tanja; Haak, Rainer; Ziebolz, Dirk

2016-07-01

The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection and Analysis of Circular RNAs by RT-PCR.

PubMed

Panda, Amaresh C; Gorospe, Myriam

2018-03-20

Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

PubMed

Klungthong, Chonticha; Manasatienkij, Wudtichai; Phonpakobsin, Thipwipha; Chinnawirotpisan, Piyawan; Rodpradit, Prinyada; Hussem, Kittinun; Thaisomboonsuk, Butsaya; Ong-ajchaowlerd, Prapapun; Nisalak, Ananda; Kalayanarooj, Siripen; Buddhari, Darunee; Gibbons, Robert V; Jarman, Richard G; Yoon, In-Kyu; Fernandez, Stefan

2015-02-01

AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods. Published by Elsevier B.V.

Targeting Mechanisms of Resistance to Taxane-Based Chemotherapy

DTIC Science & Technology

2007-09-01

gene ; monoamine oxidase A ( MAOA ) was upregulated in patients with PSA relapse (Figure 5A). Quantitative real-time PCR (qRT-PCR) was performed to...resistance and uncover mechanisms or pathways suitable for targeting with the objective of improving tumor responses to chemotherapy. Gene expression...CXCL10 but not IL8 conferring chemoresistance to prostate cancer cells. When using longer term clinical outcome, we found genes correlated with PSA

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

NASA Astrophysics Data System (ADS)

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

PubMed

Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

2016-04-25

The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia. Copyright © 2016 Elsevier B.V. All rights reserved.

MiR-422a as a Potential Cellular MicroRNA Biomarker for Postmenopausal Osteoporosis

PubMed Central

Cao, Zheng; Moore, Benjamin T.; Wang, Yang; Peng, Xian-Hao; Lappe, Joan M.; Recker, Robert R.; Xiao, Peng

2014-01-01

Background MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis. Methodology/Principal Findings We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression. Conclusions/Significance Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis. PMID:24820117

MiR-422a as a potential cellular microRNA biomarker for postmenopausal osteoporosis.

PubMed

Cao, Zheng; Moore, Benjamin T; Wang, Yang; Peng, Xian-Hao; Lappe, Joan M; Recker, Robert R; Xiao, Peng

2014-01-01

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis. We used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression. Our study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.

Development and validation of a gene profile predicting benefit of postmastectomy radiotherapy in patients with high-risk breast cancer: a study of gene expression in the DBCG82bc cohort.

PubMed

Tramm, Trine; Mohammed, Hayat; Myhre, Simen; Kyndi, Marianne; Alsner, Jan; Børresen-Dale, Anne-Lise; Sørlie, Therese; Frigessi, Arnoldo; Overgaard, Jens

2014-10-15

To identify genes predicting benefit of radiotherapy in patients with high-risk breast cancer treated with systemic therapy and randomized to receive or not receive postmastectomy radiotherapy (PMRT). The study was based on the Danish Breast Cancer Cooperative Group (DBCG82bc) cohort. Gene-expression analysis was performed in a training set of frozen tumor tissue from 191 patients. Genes were identified through the Lasso method with the endpoint being locoregional recurrence (LRR). A weighted gene-expression index (DBCG-RT profile) was calculated and transferred to quantitative real-time PCR (qRT-PCR) in corresponding formalin-fixed, paraffin-embedded (FFPE) samples, before validation in FFPE from 112 additional patients. Seven genes were identified, and the derived DBCG-RT profile divided the 191 patients into "high LRR risk" and "low LRR risk" groups. PMRT significantly reduced risk of LRR in "high LRR risk" patients, whereas "low LRR risk" patients showed no additional reduction in LRR rate. Technical transfer of the DBCG-RT profile to FFPE/qRT-PCR was successful, and the predictive impact was successfully validated in another 112 patients. A DBCG-RT gene profile was identified and validated, identifying patients with very low risk of LRR and no benefit from PMRT. The profile may provide a method to individualize treatment with PMRT. ©2014 American Association for Cancer Research.

High prevalence of asymptomatic malaria infections: a cross-sectional study in rural areas in six departments in Haiti.

PubMed

Elbadry, Maha A; Al-Khedery, Basima; Tagliamonte, Massimiliano S; Yowell, Charles A; Raccurt, Christian P; Existe, Alexandre; Boncy, Jacques; Weppelmann, Thomas A; Beau De Rochars, Valery E M; Lemoine, Jean F; Okech, Bernard A; Dame, John B

2015-12-21

Public health measures are poised for transition from malaria control to malaria elimination on the island of Hispaniola. Assessment of the reservoir of asymptomatic infections from which acute malaria cases may derive is critical to plan and evaluate elimination efforts. Current field technology is ill suited for detecting sub-microscopic infections, thus highly sensitive survey methods capable of detecting virtually all infections are needed. In this study the prevalence of infection with Plasmodium falciparum was determined in patients seeking medical care primarily for non-febrile conditions in six departments in Haiti using a newly designed qRT-PCR-based assay. Three different methods of parasite detection were compared to assess their utility in approximating the prevalence of P. falciparum infections in the population: malaria rapid diagnostic test (RDT) designed to detect histidine-rich protein 2 (HRP2), thick smear microscopy, and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based upon the small sub-unit ribosomal RNA. The limit of detection of the qRT-PCR assay utilized was 0.0003 parasite/µL of blood. Venous blood was obtained from a total of 563 subjects from six departments in Haiti, all of whom were seeking medical attention without complaints consistent with malaria. Each subject was questioned for knowledge and behaviour using demographic and epidemiological survey to identify risk factors for disease transmission. Among the 563 samples tested, ten and 16 were found positive for malaria by RDT and microscopy, respectively. Using the qRT-PCR test to assess the infection status of these subjects, an additional 92 were identified for a total of 108. Based upon the qRT-PCR assay results, a wide variation in prevalence of infection in asymptomatic subjects was seen between geographic locations ranging from 4-41%. The prevalence of infection was highest in the Grand Anse, Nord and Sud-Est Departments, and demographic data from questionnaires provide evidence for focal disease transmission. The qRT-PCR assay is sufficiently sensitive to identify an unexpectedly large number of asymptomatic, submicroscopic infections. Identifying and clearing these infections presents a significant challenge to both control and elimination efforts, but the qRT-PCR assay offers a reliable method to identify them.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Comparison of EPA Method 1615 RT-qPCR Assays in Standard and Kit Format

EPA Science Inventory

EPA Method 1615 contains protocols for measuring enterovirus and norovirus by reverse transcription quantitative polymerase chain reaction. A commercial kit based upon these protocols was designed and compared to the method's standard approach. Reagent grade, secondary effluent, ...

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

PubMed

Looi, Kevin; Troy, Niamh M; Garratt, Luke W; Iosifidis, Thomas; Bosco, Anthony; Buckley, Alysia G; Ling, Kak-Ming; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Shaw, Nicole C; Sutanto, Erika N; Zosky, Graeme R; Rigby, Paul J; Larcombe, Alexander N; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2016-10-11

No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID 50 ) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT 2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. HRV-1B infection affected viability that was both time and TCID 50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID 50 , while a significant decrease in all three TJ protein expressions occurred at higher TCID 50 . Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

PubMed

Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

2009-06-01

Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P < 0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P < 0.05). There were no significant differences in AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.

PubMed

Gruber, Kim; Horn, Heike; Kalla, Jörg; Fritz, Peter; Rosenwald, Andreas; Kohlhäufl, Martin; Friedel, Godehard; Schwab, Matthias; Ott, German; Kalla, Claudia

2014-03-01

The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK. We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry. qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

PubMed

Mogal, Ashish; Abdulkadir, Sarki A

2006-04-01

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles

PubMed Central

Bousse, Tatiana; Shore, David A.; Goldsmith, Cynthia S.; Hossain, M. Jaber; Jang, Yunho; Davis, Charles T.; Donis, Ruben O.; Stevens, James

2017-01-01

Summary Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9 %. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus has greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed. PMID:23916678

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meagher, Robert J.; Ball, Cameron Scott; Langevin, Stanley A.

In this study, collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized publicmore » health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.« less

Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification

PubMed Central

Wheeler, Sarah S.; Ball, Cameron S.; Langevin, Stanley A.; Fang, Ying; Coffey, Lark L.; Meagher, Robert J.

2016-01-01

Collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized public health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance. PMID:26807734

Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.

PubMed

Vendrame, Marco; Manzano, Marisa; Comi, Giuseppe; Bertrand, Julien; Iacumin, Lucilla

2014-09-01

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer. Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid Diagnosis of Ebola Hemorrhagic Fever by Reverse Transcription-PCR in an Outbreak Setting and Assessment of Patient Viral Load as a Predictor of Outcome

PubMed Central

Towner, Jonathan S.; Rollin, Pierre E.; Bausch, Daniel G.; Sanchez, Anthony; Crary, Sharon M.; Vincent, Martin; Lee, William F.; Spiropoulou, Christina F.; Ksiazek, Thomas G.; Lukwiya, Mathew; Kaducu, Felix; Downing, Robert; Nichol, Stuart T.

2004-01-01

The largest outbreak on record of Ebola hemorrhagic fever (EHF) occurred in Uganda from August 2000 to January 2001. The outbreak was centered in the Gulu district of northern Uganda, with secondary transmission to other districts. After the initial diagnosis of Sudan ebolavirus by the National Institute for Virology in Johannesburg, South Africa, a temporary diagnostic laboratory was established within the Gulu district at St. Mary's Lacor Hospital. The laboratory used antigen capture and reverse transcription-PCR (RT-PCR) to diagnose Sudan ebolavirus infection in suspect patients. The RT-PCR and antigen-capture diagnostic assays proved very effective for detecting ebolavirus in patient serum, plasma, and whole blood. In samples collected very early in the course of infection, the RT-PCR assay could detect ebolavirus 24 to 48 h prior to detection by antigen capture. More than 1,000 blood samples were collected, with multiple samples obtained from many patients throughout the course of infection. Real-time quantitative RT-PCR was used to determine the viral load in multiple samples from patients with fatal and nonfatal cases, and these data were correlated with the disease outcome. RNA copy levels in patients who died averaged 2 log10 higher than those in patients who survived. Using clinical material from multiple EHF patients, we sequenced the variable region of the glycoprotein. This Sudan ebolavirus strain was not derived from either the earlier Boniface (1976) or Maleo (1979) strain, but it shares a common ancestor with both. Furthermore, both sequence and epidemiologic data are consistent with the outbreak having originated from a single introduction into the human population. PMID:15047846

Development and validation of a gene expression oligo microarray for the gilthead sea bream (Sparus aurata).

PubMed

Ferraresso, Serena; Vitulo, Nicola; Mininni, Alba N; Romualdi, Chiara; Cardazzo, Barbara; Negrisolo, Enrico; Reinhardt, Richard; Canario, Adelino V M; Patarnello, Tomaso; Bargelloni, Luca

2008-12-03

Aquaculture represents the most sustainable alternative of seafood supply to substitute for the declining marine fisheries, but severe production bottlenecks remain to be solved. The application of genomic technologies offers much promise to rapidly increase our knowledge on biological processes in farmed species and overcome such bottlenecks. Here we present an integrated platform for mRNA expression profiling in the gilthead sea bream (Sparus aurata), a marine teleost of great importance for aquaculture. A public data base was constructed, consisting of 19,734 unique clusters (3,563 contigs and 16,171 singletons). Functional annotation was obtained for 8,021 clusters. Over 4,000 sequences were also associated with a GO entry. Two 60mer probes were designed for each gene and in-situ synthesized on glass slides using Agilent SurePrint technology. Platform reproducibility and accuracy were assessed on two early stages of sea bream development (one-day and four days old larvae). Correlation between technical replicates was always > 0.99, with strong positive correlation between paired probes. A two class SAM test identified 1,050 differentially expressed genes between the two developmental stages. Functional analysis suggested that down-regulated transcripts (407) in older larvae are mostly essential/housekeeping genes, whereas tissue-specific genes are up-regulated in parallel with the formation of key organs (eye, digestive system). Cross-validation of microarray data was carried out using quantitative qRT-PCR on 11 target genes, selected to reflect the whole range of fold-change and both up-regulated and down-regulated genes. A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates. Good concordance between qRT-PCR and microarray data was observed between 2- and 7-fold change, while fold-change compression in the microarray was present for differences greater than 10-fold in the qRT-PCR. A highly reliable oligo-microarray platform was developed and validated for the gilthead sea bream despite the presently limited knowledge of the species transcriptome. Because of the flexible design this array will be able to accommodate additional probes as soon as novel unique transcripts are available.

Fast-mode duplex qPCR for BCR-ABL1 molecular monitoring: innovation, automation, and harmonization.

PubMed

Gerrard, Gareth; Mudge, Katherine; Foskett, Pierre; Stevens, David; Alikian, Mary; White, Helen E; Cross, Nicholas C P; Apperley, Jane; Foroni, Letizia

2012-07-01

Reverse transcription quantitative polymerase chain reaction (RTqPCR)is currently the most sensitive tool available for the routine monitoring of disease level in patients undergoing treatment for BCRABL1 associated malignancies. Considerable effort has been invested at both the local and international levels to standardise the methodology and reporting criteria used to assess this critical metric. In an effort to accommodate the demands of increasing sample throughput and greater standardization, we adapted the current best-practice guidelines to encompass automation platforms and improved multiplex RT-qPCR technology.

[Comparative cost analysis of molecular biology methods in the diagnosis of sarcomas].

PubMed

Baffert, Sandrine; Italiano, Antoine; Pierron, Gaëlle; Traoré, Marie-Angèle; Rapp, Jocelyn; Escande, Fabienne; Ghnassia, Jean-Pierre; Terrier, Philippe; Voegeli, Anne-Claire; Ranchere-Vince, Dominique; Coindre, Jean-Michel; Pedeutour, Florence

2013-10-01

Sarcomas represent a complex and heterogeneous group of rare malignant tumors and their correct diagnosis is often difficult. Recent molecular biological techniques have been of great diagnostic use and there is a need to assess the cost of these procedures in routine clinical practice. Using prospective and observational data from eight molecular biology laboratories in France, we used "microcosting" method to assess the cost of molecular biological techniques in the diagnosis of five types of sarcoma. The mean cost of fluorescence in situ hybridization (FISH) was 318 € (273-393) per sample; mean reverse transcription polymerase chain reaction (RT-PCR) cost ranged from 300 € (229-481) per formalin-fixed, paraffin-embedded specimen to 258 € (213-339) per frozen specimen; mean quantitative polymerase chain reaction (Q-PCR) cost was 184 € (112-229) and mean CGH-array cost was 332 € (329-335). The cost of these recently implemented techniques varied according to the type of sarcoma; the method of tissue collection and local organizational factors including the level of local expertise and investment. The cost of molecular diagnostic techniques needs to be balanced against their respective performance.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA.

PubMed

James, Amanda Marie; Baker, Meredith B; Bao, Gang; Searles, Charles D

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro . The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA

PubMed Central

James, Amanda Marie; Baker, Meredith B.; Bao, Gang; Searles, Charles D.

2017-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples. PMID:28255356

Concentration of Enteroviruses, Adenoviruses, and Noroviruses from Drinking Water by Use of Glass Wool Filters▿

PubMed Central

Lambertini, Elisabetta; Spencer, Susan K.; Bertz, Phillip D.; Loge, Frank J.; Kieke, Burney A.; Borchardt, Mark A.

2008-01-01

Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min−1), and its low cost makes it advantageous for studies requiring large numbers of samples. PMID:18359827

[miR-25 promotes cell proliferation by targeting RECK in human cervical carcinoma HeLa cells].

PubMed

Qiu, Gang; Fang, Baoshuan; Xin, Guohong; Wei, Qiang; Yuan, Xiaoye; Wu, Dayong

2015-01-01

To investigate the effect of miR-25 on the proliferation of human cervical carcinoma HeLa cells and its association with reversion-inducing cysteine-rich protein with Kazal motifs (RECK). The recombinant plasmids of pcDNATM6.2-GW-pre-miR-25, pmirGLO-RECK-WT, pmirGLO-RECK-MT and anti-miR-25 were constructed, and their transfection efficiencies into HeLa cells were identified by real-time quantitative PCR (qRT-PCR). The potential proliferation-stimulating function of miR-25 was analyzed by MTT assay in HeLa cells. Furthermore, the target effect of miR-25 on the RECK was determined by dual-luciferase reporter assay system, qRT-PCR and Western blotting. Sequence analysis demonstrated that the recombinant plasmids of pcDNATM6.2-GW-pre-miR-25 and pmirGLO-RECK-WT, pmirGLO-RECK-MT were successfully constructed, and qRT-PCR revealed that the transfection efficiencies of pre-miR-25 and anti-miR-25 were desirable in HeLa cells. MTT assay showed that miR-25 over-expression promoted the proliferation of HeLa cells. In addition, the luciferase activity was significantly reduced in HeLa cells cotransfected with pre-miR-25 and RECK-WT. The qRT-PCR and Western blotting indicated that the expression level of RECK was up-regulated in HeLa cells transfected with anti-miR-25 at the transcriptional and posttranscriptional levels. miR-25 could promote cell proliferation by targeting RECK in HeLa cells.

Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

PubMed Central

Chouteau, Mathieu; Whibley, Annabel; Joron, Mathieu; Llaurens, Violaine

2016-01-01

Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. PMID:27271971

Induction of a Pregnancy-Like Mammary Gland Differentiation by Docosapentaenoic Omega-3 Fatty Acid

DTIC Science & Technology

2008-09-01

xylenes, and stored in methyl salicylate . Morphological Assessment of Mammary Gland—Whole inguinal mammary glands were removed from virgin control as...respectively, defatted in xylenes, and stored in methyl salicylate . Quantitative RT-PCR analyses RNA was isolated and subjected to real time PCR analysis... methylation , and fatty acid analysis were performed as previously described [28,48]. Briefly, an ali- quot of mammary tissue homogenate in a glass

Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

PubMed

Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

2005-09-01

1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

PubMed

Cai, Jing; Li, Pengfei; Luo, Xiao; Chang, Tianliang; Li, Jiaxing; Zhao, Yuwei; Xu, Yao

2018-01-01

Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.

PubMed

Kim, Y H; Cho, K W; Youn, H Y; Yoo, H S; Han, H R

2001-04-01

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

High-throughput deep screening and identification of four peripheral leucocyte microRNAs as novel potential combination biomarkers for preeclampsia

PubMed Central

Wang, Yonghong; Yang, Xukui; Yang, Yuanyuan; Wang, Wenjun; Zhao, Meiling; Liu, Huiqiang; Li, Dongyan; Hao, Min

2016-01-01

Objective: To identify the specific microRNA (miRNA) biomarkers of preeclampsia (PE), the miRNA profiles analysis were performed. Study Design: The blood samples were obtained from five PE patients and five normal healthy pregnant women. The small RNA profiles were analyzed to identify miRNA expression levels and find out miRNAs that may associate with PE. The quantitative reverse transcriptase–PCR (qRT-PCR) assay was used to validate differentially expressed peripheral leucocyte miRNAs in a new cohort. Result: The data analysis showed that 10 peripheral leucocyte miRNAs were significantly differently expressed in severe PE patients. Four differently expressed miRNAs were successfully validated using qRT-PCR method. Conclusion: We successfully constructed a model with high accuracy to predict PE. A combination of four peripheral leucocyte miRNAs has great potential to serve as diagnostic biomarkers of PE. PMID:26675000

Quantitative analysis of hepatitis C virus activity in vivo in different groups of untreated patients.

PubMed

Manzin, A; Solforosi, L; Giostra, F; Bianchi, F B; Bruno, S; Rossi, S; Gabrielli, A; Candela, M; Petrelli, E; Clementi, M

1997-01-01

Highly sensitive competitive PCR (cPCR) and competitive reverse transcription PCR (cRT-PCR) methodologies were recently developed and applied for quantifying viral DNA and RNA species (including HCV RNA) present in clinical samples at low concentration. In this study, we used cRT-PCR to compare the viral load of 118 untreated patients with HCV infection and different clinical conditions (80 patients with chronic hepatitis, 18 infected subjects with persistently normal ALT levels and various degrees of liver injury, 10 HCV infected subjects that tested positive for anti-LKM1 antibodies, and 10 patients with HCV infection and cryoglobulinemia). The results indicate that while great individual variability of HCV viremia is detectable even among patients with similar clinical conditions, the mean HCV RNA copy number in samples from patients with different clinical conditions was similar in all groups with the single exception of patients that tested positive for anti-liver-kidney microsomal auto-antibodies type 1 (anti-LKM1); interestingly, lower HCV viremia levels were revealed in these anti-LKM1-positive cases with liver disease of uncertain pathogenesis.

Changes in content and synthesis of collagen types and proteoglycans in osteoarthritis of the knee joint and comparison of quantitative analysis with Photoshop-based image analysis.

PubMed

Lahm, Andreas; Mrosek, Eike; Spank, Heiko; Erggelet, Christoph; Kasch, Richard; Esser, Jan; Merk, Harry

2010-04-01

The different cartilage layers vary in synthesis of proteoglycan and of the distinct types of collagen with the predominant collagen Type II with its associated collagens, e.g. types IX and XI, produced by normal chondrocytes. It was demonstrated that proteoglycan decreases in degenerative tissue and a switch from collagen type II to type I occurs. The aim of this study was to evaluate the correlation of real-time (RT)-PCR and Photoshop-based image analysis in detecting such lesions and find new aspects about their distribution. We performed immunohistochemistry and histology with cartilage tissue samples from 20 patients suffering from osteoarthritis compared with 20 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. Using Adobe Photoshop the digitized images of histology and immunohistochemistry stains of collagen type I and II were stored on an external data storage device. The area occupied by any specific colour range can be specified and compared in a relative manner directly from the histogram using the "magic wand tool" in the select similar menu. In the image grow menu gray levels or luminosity (colour) of all pixels within the selected area, including mean, median and standard deviation, etc. are depicted. Statistical Analysis was performed using the t test. With the help of immunohistochemistry, RT-PCR and quantitative RT- PCR we found that not only collagen type II, but also collagen type I is synthesized by the cells of the diseased cartilage tissue, shown by increasing amounts of collagen type I mRNA especially in the later stages of osteoarthritis. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which leads to an overall decrease. Analysis of proteoglycan showed a loss of the overall content and a quite uniform staining in the different zones compared to the healthy cartilage with a classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR using Spearman correlation analysis revealed strong correlation for Safranin O and collagen type I, medium for collagen type II and glycoprotein but weak correlation between PCR aggrecan results. Photoshop-based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular cartilage.

Colorimetric TMPRSS2-ERG Gene Fusion Detection in Prostate Cancer Urinary Samples via Recombinase Polymerase Amplification.

PubMed

Koo, Kevin M; Wee, Eugene J H; Trau, Matt

2016-01-01

TMPRSS2 (Exon 1)-ERG (Exon 4) is the most frequent gene fusion event in prostate cancer (PC), and is highly PC-specific unlike the current serum prostate specific antigen (PSA) biomarker. However, TMPRSS2-ERG levels are currently measured with quantitative reverse-transcription PCR (RT-qPCR) which is time-consuming and requires costly equipment, thus limiting its use in clinical diagnostics. Herein, we report a novel rapid, cost-efficient and minimal-equipment assay named "FusBLU" for detecting TMPRSS2-ERG gene fusions from urine. TMPRSS2-ERG mRNA was amplified by isothermal reverse transcription-recombinase polymerase amplification (RT-RPA), magnetically-isolated, and detected through horseradish peroxidase (HRP)-catalyzed colorimetric reaction. FusBLU was specific for TMPRSS2-ERG mRNA with a low visual detection limit of 10(5) copies. We also demonstrated assay readout versatility on 3 potentially useful platforms. The colorimetric readout was detectable by naked eye for a quick yes/no evaluation of gene fusion presence. On the other hand, a more quantitative TMPRSS2-ERG detection was achievable by absorbance/electrochemical measurements. FusBLU was successfully applied to 12 urinary samples and results were validated by gold-standard RT-qPCR. We also showed that sediment RNA was likely the main source of TMPRSS2-ERG mRNA in urinary samples. We believe that our assay is a potential clinical screening tool for PC and could also have wide applications for other disease-related fusion genes.

Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

PubMed Central

Pichler, Martin; Zatloukal, Kurt

2013-01-01

Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples. PMID:23936242

Presenilins in the heart: presenilin-2 expression is increased by low glucose and by hypoxia in cardiac cells.

PubMed

Mohuczy, Dagmara; Qian, Keping; Phillips, M Ian

2002-12-31

Cardiac cells are subjected to hypoxia in many cardiovascular diseases. We studied a broad spectrum of genes using a macroarrays-based method to analyze RNA of rat cardiac fetal cell line H9c2 after 4 h of hypoxic conditions in the incubator-1% oxygen concentration, as compared to normoxic conditions (21% oxygen). The cDNAs were prepared from total RNAs using Atlas Rat 1.2 Array (Clontech Laboratories) and hybridized to the membrane containing 1176 rat cDNAs and 9 housekeeping control cDNAs. Genes expression was analyzed using AtlasImage 1.01 software. We found over 45 genes up-regulated in a range of 1.5-2.9 times and 9 genes down-regulated to a range of 0.4-0.7 times, under hypoxia versus normoxia. Presenilin-2 (PS2) was detected in the cultured heart cells. RT-PCR confirmed the presence of PS2 in the heart of adult rats. Using quantitative real-time RT-PCR, we further studied the expression of presenilin-2 mRNA under conditions of low oxygen supply and glucose starvation. Glucose deprivation itself caused significant up-regulation of the presenilin-2 (to 160%) and with low oxygen increased presenilin-2 level to over 200% of the control. Presenilin-2 has previously been associated with intercellular signaling in the central nervous system, in Alzheimer's disease. The finding of presenilin-2 in the heart and the responsiveness to low glucose and hypoxia suggests that PS2 may be regulated by conditions of ischemia, a condition which both the heart and brain may experience.

A novel X-linked disorder with developmental delay and autistic features.

PubMed

Kaya, Namik; Colak, Dilek; Albakheet, Albandary; Al-Owain, Mohammad; Abu-Dheim, Nada; Al-Younes, Banan; Al-Zahrani, Jawaher; Mukaddes, Nahit M; Dervent, Aysin; Al-Dosari, Naji; Al-Odaib, Ali; Kayaalp, Inci V; Al-Sayed, Moeenaladin; Al-Hassnan, Zuhair; Nester, Michael J; Al-Dosari, Mohammad; Al-Dhalaan, Hesham; Chedrawi, Aziza; Gunoz, Hulya; Karakas, Bedri; Sakati, Nadia; Alkuraya, Fowzan S; Gascon, Generaso G; Ozand, Pinar T

2012-04-01

Genomic duplications that lead to autism and other human diseases are interesting pathological lesions since the underlying mechanism almost certainly involves dosage sensitive genes. We aim to understand a novel genomic disorder with profound phenotypic consequences, most notably global developmental delay, autism, psychosis, and anorexia nervosa. We evaluated the affected individuals, all maternally related, using childhood autism rating scale (CARS) and Vineland Adaptive scales, magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) brain, electroencephalography (EEG), electromyography (EMG), muscle biopsy, high-resolution molecular karyotype arrays, Giemsa banding (G-banding) and fluorescent in situ hybridization (FISH) experiments, mitochondrial DNA (mtDNA) sequencing, X-chromosome inactivation study, global gene expression analysis on Epstein-Barr virus (EBV)-transformed lymphoblasts, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We have identified a novel Xq12-q13.3 duplication in an extended family. Clinically normal mothers were completely skewed in favor of the normal chromosome X. Global transcriptional profiling of affected individuals and controls revealed significant alterations of genes and pathways in a pattern consistent with previous microarray studies of autism spectrum disorder patients. Moreover, expression analysis revealed copy number-dependent increased messenger RNA (mRNA) levels in affected patients compared to control individuals. A subset of differentially expressed genes was validated using qRT-PCR. Xq12-q13.3 duplication is a novel global developmental delay and autism-predisposing chromosomal aberration; pathogenesis of which may be mediated by increased dosage of genes contained in the duplication, including NLGN3, OPHN1, AR, EFNB1, TAF1, GJB1, and MED12. Copyright © 2011 American Neurological Association.

MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

PubMed Central

Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan; Abdallah, Basem M.; Ditzel, Nicholas; Nossent, Anne Yael; Bak, Mads; Kauppinen, Sakari; Kassem, Moustapha

2011-01-01

Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo. PMID:21444814

Screening of lymph nodes metastasis associated lncRNAs in colorectal cancer patients

PubMed Central

Han, Jun; Rong, Long-Fei; Shi, Chuan-Bin; Dong, Xiao-Gang; Wang, Jie; Wang, Bao-Lin; Wen, Hao; He, Zhen-Yu

2014-01-01

AIM: To screen lymph nodes metastasis associated long noncoding RNAs (lncRNAs) in colorectal cancer through microarray analysis. METHODS: Metastatic lymph node (MLN), normal lymph node (NLN) and tumor tissues of 3 colorectal cancer (CRC) patients were collected during the operation and validated by pathological examinations. RNAs were extracted from MLN, NLN, and cancer tissues separately. RNA quantity and quality were measured with a NanoDrop ND-1000 spectrophotometer and RNA integrity was assessed by standard denaturing agarose electrophoresis. Agilent Feature Extraction Software (Version 11.0.1.1) was used to analyze acquired array images. Four differently expressed lncRNAs were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) in 26 subsets of MLN, NLN, and tumor tissues. RESULTS: Of 33045 lncRNAs, 1133 were differentially expressed in MLN compared with NLN, of which 260 were up-regulated and 873 down-regulated (≥ 2 fold-change). Five hundred and forty-five lncRNAs were differentially expressed in MLN compared with tumor tissues, of which 460 were up-regulated and 85 down-regulated (≥ 2 fold-change). Compared with NLN and cancer tissues, 14 lncRNAs were specifically up-regulated and 5 specifically down-regulated in MLN. AK307796, ENST00000425785, and AK021444 were confirmed to be specifically up-regulated in MLN and ENST00000465846 specifically down-regulated in MLN by qRT-PCR in 26 CRC patients. CONCLUSION: The specifically expressed lncRNAs in MLN may exert a partial or key role in the progress of lymph nodes metastasis of CRC. PMID:25009386

Intra-Platform Repeatability and Inter-Platform Comparability of MicroRNA Microarray Technology

PubMed Central

Sato, Fumiaki; Tsuchiya, Soken; Terasawa, Kazuya; Tsujimoto, Gozoh

2009-01-01

Over the last decade, DNA microarray technology has provided a great contribution to the life sciences. The MicroArray Quality Control (MAQC) project demonstrated the way to analyze the expression microarray. Recently, microarray technology has been utilized to analyze a comprehensive microRNA expression profiling. Currently, several platforms of microRNA microarray chips are commercially available. Thus, we compared repeatability and comparability of five different microRNA microarray platforms (Agilent, Ambion, Exiqon, Invitrogen and Toray) using 309 microRNAs probes, and the Taqman microRNA system using 142 microRNA probes. This study demonstrated that microRNA microarray has high intra-platform repeatability and comparability to quantitative RT-PCR of microRNA. Among the five platforms, Agilent and Toray array showed relatively better performances than the others. However, the current lineup of commercially available microRNA microarray systems fails to show good inter-platform concordance, probably because of lack of an adequate normalization method and severe divergence in stringency of detection call criteria between different platforms. This study provided the basic information about the performance and the problems specific to the current microRNA microarray systems. PMID:19436744

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

PubMed Central

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna MY; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease. PMID:26011746

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

PubMed

Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

2015-01-01

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

Genome-wide analysis of SINA family in plants and their phylogenetic relationships.

PubMed

Wang, Meng; Jin, Ying; Fu, Junjie; Zhu, Yun; Zheng, Jun; Hu, Jian; Wang, Guoying

2008-06-01

SINA genes in plants are part of a multigene family with 5 members in Arabidopsis thaliana, 10 members in Populus trichocarpa, 6 members in Oryza sativa, at least 6 members in Zea mays and at least 1 member in Physcomitrella patens. Six members in maize were confirmed by RT-PCR. All SINAs have one RING domain and one SINA domain. These two domains are highly conserved in plants. According to the motif organization and phylogenetic tree, SINA family members were divided into 2 groups. In addition, through semi-quantitative RT-PCR analysis of maize members and Digital Northern analysis of Arabidopsis and rice members, we found that the tissue expression patterns are more diverse in monocot than in Arabidopsis.

[Fluorescence quantitative PCR detection of WT1 gene expression in peripheral blood of patients with acute leukemias and its clinical implications].

PubMed

Bai, Bo; Wang, Hong-Wei; Xu, Yong-Qun; Yang, Hei-Nu; Qiao, Zhen-Hua

2005-08-01

To elucidate the expression of WT1 in all types of leukemias and its implications for monitoring minimal residual disease in patients with acute leukemia, the peripheral blood from 55 leukemia patients and 10 normal voluteer was detected by using FQ-RT-PCR. Follow-up monitoring of WT1 expression of peripheral blood was performed for 20 patients with acute leukemia. The results showed that the expression of WT1 gene in all types of leukemias was significantly higher than that in normal control (P < 0.001). For ANLL and ALL patients, the survival time in the group of WT1 6.8 x 10(-3), (P = 0.027). Follow-up detection of the expression of WT1 in peripheral blood samples from 20 acute leukemia patients, 7 cases relapsed after complete remission has been done. In 5 of 7 relapsed patients, the expression of WT1 had obviously increased about 2 - 3 months before clinical relapse became apparent. It is concluded that the established FQ-RT-PCR method is accurate and specific. The expression of WT1 gene is relatively high in all types of leukemias compared with normal peripheral blood cells, the higher WT1 expression may associate with poor prognosis in acute leukemia, and the dynamics of WT1 level correlate with the disease status. The quantitative assessment of WT1 expression in peripheral blood samples by FQ-RT-PCR may be a useful tool for monitoring minimal residual disease.

Evaluation and Selection of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Withania somnifera (L.) Dunal

PubMed Central

Singh, Varinder; Kaul, Sunil C.; Wadhwa, Renu; Pati, Pratap Kumar

2015-01-01

Quantitative real-time PCR (qRT-PCR) is now globally used for accurate analysis of transcripts levels in plants. For reliable quantification of transcripts, identification of the best reference genes is a prerequisite in qRT-PCR analysis. Recently, Withania somnifera has attracted lot of attention due to its immense therapeutic potential. At present, biotechnological intervention for the improvement of this plant is being seriously pursued. In this background, it is important to have comprehensive studies on finding suitable reference genes for this high valued medicinal plant. In the present study, 11 candidate genes were evaluated for their expression stability under biotic (fungal disease), abiotic (wounding, salt, drought, heat and cold) stresses, in different plant tissues and in response to various plant growth regulators (methyl jasmonate, salicylic acid, abscisic acid). The data as analyzed by various software packages (geNorm, NormFinder, Bestkeeper and ΔCt method) suggested that cyclophilin (CYP) is a most stable gene under wounding, heat, methyl jasmonate, different tissues and all stress conditions. T-SAND was found to be a best reference gene for salt and salicylic acid (SA) treated samples, while 26S ribosomal RNA (26S), ubiquitin (UBQ) and beta-tubulin (TUB) were the most stably expressed genes under drought, biotic and cold treatment respectively. For abscisic acid (ABA) treated samples 18S-rRNA was found to stably expressed gene. Finally, the relative expression level of the three genes involved in the withanolide biosynthetic pathway was detected to validate the selection of reliable reference genes. The present work will significantly contribute to gene analysis studies in W. somnifera and facilitate in improving the quality of gene expression data in this plant as well as and other related plant species. PMID:25769035

Application of Reverse Transcriptase -PCR (RT-PCR) for rapid detection of viable Escherichia coli in drinking water samples.

PubMed

Molaee, Neda; Abtahi, Hamid; Ghannadzadeh, Mohammad Javad; Karimi, Masoude; Ghaznavi-Rad, Ehsanollah

2015-01-01

Polymerase chain reaction (PCR) is preferred to other methods for detecting Escherichia coli (E. coli) in water in terms of speed, accuracy and efficiency. False positive result is considered as the major disadvantages of PCR. For this reason, reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to solve this problem. The aim of present study was to determine the efficiency of RT-PCR for rapid detection of viable Escherichia coli in drinking water samples and enhance its sensitivity through application of different filter membranes. Specific primers were designed for 16S rRNA and elongation Factor II genes. Different concentrations of bacteria were passed through FHLP and HAWP filters. Then, RT-PCR was performed using 16srRNA and EF -Tu primers. Contamination of 10 wells was determined by RT-PCR in Arak city. To evaluate RT-PCR efficiency, the results were compared with most probable number (MPN) method. RT-PCR is able to detect bacteria in different concentrations. Application of EF II primers reduced false positive results compared to 16S rRNA primers. The FHLP hydrophobic filters have higher ability to absorb bacteria compared with HAWB hydrophilic filters. So the use of hydrophobic filters will increase the sensitivity of RT-PCR. RT-PCR shows a higher sensitivity compared to conventional water contamination detection method. Unlike PCR, RT-PCR does not lead to false positive results. The use of EF-Tu primers can reduce the incidence of false positive results. Furthermore, hydrophobic filters have a higher ability to absorb bacteria compared to hydrophilic filters.

Specific metabolic activity of ripening bacteria quantified by real-time reverse transcription PCR throughout Emmental cheese manufacture.

PubMed

Falentin, Hélène; Postollec, Florence; Parayre, Sandrine; Henaff, Nadine; Le Bivic, Pierre; Richoux, Romain; Thierry, Anne; Sohier, Danièle

2010-11-15

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 10(3) copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

PubMed

Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

2015-03-01

Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

Validation of high-throughput single cell analysis methodology.

PubMed

Devonshire, Alison S; Baradez, Marc-Olivier; Morley, Gary; Marshall, Damian; Foy, Carole A

2014-05-01

High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection. We compared protocols using a separate lysis buffer with cell capture directly in RT-PA reagent. The two methods were found to have similar lysis efficiencies, whereas the direct RT-PA approach showed improved precision. Digital PCR was used to relate preamplified template copy numbers to Cq values and reveal where low-quality signals may affect the analysis. We investigated the impact of calibration and data normalization strategies as a means of minimizing the impact of inter-experimental variation on gene expression values and found that both approaches can improve data comparability. This study provides validation and guidance for the application of high-throughput qPCR workflows for gene expression profiling of single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Isolation and characterization of a FLOWERING LOCUS T homolog from pineapple (Ananas comosus (L.) Merr).

PubMed

Lv, LingLing; Duan, Jun; Xie, JiangHui; Wei, ChangBin; Liu, YuGe; Liu, ShengHui; Sun, GuangMing

2012-09-01

FLOWERING LOCUS T (FT)-like genes are crucial regulators of flowering in angiosperms. A homolog of FT, designated as AcFT (GenBank ID: HQ343233), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcFT is 915 bp in length and contains an ORF of 534 bp, which encodes a protein of 177 aa. Molecular weight was 19.9 kDa and isoelectric point was 6.96. The deduced protein sequence of AcFT was 84% and 82% identical to homologs encoded by CgFT in Cymbidium goeringii and OgFT in Oncidium Gower Ramsey respectively. Quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AcFT was high in flesh and none in leaves. qRT-PCR analyses in different stages indicated that the expression of AcFT reached the highest level on 40 d after flower inducing, when the multiple fruit and floral organs were forming. The 35S::AcFT transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Clearance of a persistent Picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

USDA-ARS?s Scientific Manuscript database

Immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in distinct microanatomic compartments of the bovine nasopharynx were investigated using quantitative RT-PCR and whole transcriptome microarray. Analysis of tissue samples obtained during ...

Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

USGS Publications Warehouse

Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

2000-01-01

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

PubMed

Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

2016-11-01

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Selection of housekeeping genes for use in quantitative reverse transcription PCR assays on the murine cornea.

PubMed

Ren, Shengwei; Zhang, Feng; Li, Changyou; Jia, Changkai; Li, Siyuan; Xi, Haijie; Zhang, Hongbo; Yang, Lingling; Wang, Yiqiang

2010-06-11

To evaluate the suitability of common housekeeping genes (HKGs) for use in quantitative reverse transcription PCR (qRT-PCR) assays of the cornea in various murine disease models. CORNEAL DISEASE MODELS STUDIED WERE: 1) corneal neovascularization (CorNV) induced by suture or chemical burn, 2) corneal infection with Candida albicans or Aspergillus fumigatus by intrastromal injection of live spores, and 3) perforating corneal injury (PCI) in Balb/c mice or C57BL/6 mice. Expression of 8 HKGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], beta-actin [ACTB], lactate dehydrogenase A [LDHA], ribosomal protein L5 [RPL5], ubiquitin C [UBC], peptidylprolyl isomerase A [PPIA], TATA-box binding protein [TBP1], and hypoxanthine guanine phosphoribosyl transferase [HPRT1]) in the cornea were measured at various time points by microarray hybridization or qRT-PCR and the data analyzed using geNorm and NormFinder. Microarray results showed that under the CorNV condition the expression stability of the 8 HKGs decreased in order of PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA. qRT-PCR analyses demonstrated that expression of none of the 8 HKGs remained stable under all conditions, while GAPDH and ACTB were among the least stably expressed markers under most conditions. Both geNorm and NormFinder analyses proposed best HKGs or HKG combinations that differ between the various models. NormFinder proposed PPIA as best HKG for three CorNV models and PCI model, as well as UBC for two fungal keratitis models. geNorm analysis demonstrated that a similar model in different mice strains or caused by different stimuli may require different HKGs or HKG pairs for the best normalization. Namely, geNorm proposed PPIA and HRPT1 and PPIA and RPL5 pairs for chemical burn-induced CorNV in Balb/c and C57BL/6 mice, respectively, while UBC and HPRT1 and UBC and LDHA were best for Candida and Aspergillus induced keratitis in Balb/c mice, respectively. When qRT-PCR is designed for studies of gene expression in murine cornea, preselection of situation-specific reference genes is recommended. In the absence of knowledge about situation-specific HKGs, PPIA and UBC, either alone or in combination with HPRT1 or RPL5, can be employed.

WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

NASA Technical Reports Server (NTRS)

Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Schonfeld, Julie

2014-01-01

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015.

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

PubMed

Kilpatrick, David R; Yang, Chen-Fu; Ching, Karen; Vincent, Annelet; Iber, Jane; Campagnoli, Ray; Mandelbaum, Mark; De, Lina; Yang, Su-Ju; Nix, Allan; Kew, Olen M

2009-06-01

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis.

PubMed

Lee, Hwanyong; Jung, Jinhee; Han, Song-I; Han, Ki-Ho

2010-10-21

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h(-1), and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (A(260)/A(280)) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.

Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

PubMed Central

Arif, M.; Aguilar-Moreno, G. S.; Wayadande, A.; Fletcher, J.

2014-01-01

A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods—real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)—for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5′ terminus, raising the primers' melting temperature (Tm; ca. 10°C) to make them compatible with RT-HDA (required optimal Tm = 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications. PMID:24162574

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

PubMed

Wilkes, Rebecca P; Tsai, Yun-Long; Lee, Pei-Yu; Lee, Fu-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2014-09-09

Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

PubMed Central

Maheux, Andrée F.; Bérubé, Ève; Boudreau, Dominique K.; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc

2013-01-01

We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP−/rtPCR+ colonies were identified as C. perfringens, whereas 3 mCP+/rtPCR− colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection. PMID:24077714

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

PubMed

Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

2015-12-01

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

Scientist | Center for Cancer Research

Cancer.gov

KEY ROLES/RESPONSIBILITIES The Scientist I will support research efforts to define the role of transcriptional regulators in myeloid cell development, and their potential role in leukemogenesis.  This work will be accomplished performing both molecular and stem cell biology techniques, cloning and construction of retroviral vectors, quantitative RT-PCR, cloning of conditional

Evaluation of isolation methods for bacterial RNA quantitation in Dickeya dadantii

USDA-ARS?s Scientific Manuscript database

Dickeya dadantii is a difficult source for RNA of a sufficient quality for real-time qRT-PCR analysis of gene expression. Three RNA isolation methods were evaluated for their ability to produce high-quality RNA from this bacterium. Bacterial lysis with Trizol using standard protocols consistently ga...

HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

PubMed

Dailey, Deanna D; Anfinsen, Kristin P; Pfaff, Liza E; Ehrhart, E J; Charles, J Brad; Bønsdorff, Tina B; Thamm, Douglas H; Powers, Barbara E; Jonasdottir, Thora J; Duval, Dawn L

2013-07-01

Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI. Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

PubMed

Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

2008-09-01

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections.

PubMed

Das, Smita; Jang, Ihn Kyung; Barney, Becky; Peck, Roger; Rek, John C; Arinaitwe, Emmanuel; Adrama, Harriet; Murphy, Maxwell; Imwong, Mallika; Ling, Clare L; Proux, Stephane; Haohankhunnatham, Warat; Rist, Melissa; Seilie, Annette M; Hanron, Amelia; Daza, Glenda; Chang, Ming; Nakamura, Tomoka; Kalnoky, Michael; Labarre, Paul; Murphy, Sean C; McCarthy, James S; Nosten, Francois; Greenhouse, Bryan; Allauzen, Sophie; Domingo, Gonzalo J

2017-11-01

Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum -induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections

PubMed Central

Das, Smita; Jang, Ihn Kyung; Barney, Becky; Peck, Roger; Rek, John C.; Arinaitwe, Emmanuel; Adrama, Harriet; Murphy, Maxwell; Imwong, Mallika; Ling, Clare L.; Proux, Stephane; Haohankhunnatham, Warat; Rist, Melissa; Seilie, Annette M.; Hanron, Amelia; Daza, Glenda; Chang, Ming; Nakamura, Tomoka; Kalnoky, Michael; Labarre, Paul; Murphy, Sean C.; McCarthy, James S.; Nosten, Francois; Greenhouse, Bryan; Allauzen, Sophie; Domingo, Gonzalo J.

2017-01-01

Abstract. Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies. PMID:28820709

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

PubMed Central

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

PubMed

De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

2018-04-01

Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

Gene expression patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suite of model toxicants

PubMed Central

Hook, Sharon E.; Skillman, Ann D.; Small, Jack A.; Schultz, Irvin R.

2008-01-01

The increased availability and use of DNA microarrays has allowed the characterization of gene expression patterns associated with exposure to different toxicants. An important question is whether toxicant induced changes in gene expression in fish are sufficiently diverse to allow for identification of specific modes of action and/or specific contaminants. In theory, each class of toxicant may generate a gene expression profile unique to its mode of toxic action. In this study, isogenic (cloned) rainbow trout Oncorhynchus mykiss were exposed to sublethal levels of a series of model toxicants with varying modes of action, including ethynylestradiol (xeno-estrogen), 2,2,4,4′-tetrabromodiphenyl ether (BDE-47, thyroid active), diquat (oxidant stressor), chromium VI, and benzo[a]pyrene (BaP) for a period of 1–3 weeks. An additional experiment measured trenbolone (anabolic steroid; model androgen) induced gene expression changes in sexually mature female trout. Following exposure, fish were euthanized, livers removed and RNA extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNA’s. The slides were scanned to measure abundance of a given transcript in each sample relative to controls. Data were analyzed via Genespring (Silicon Genetics) to identify a list of up- and downregulated genes, as well as to determine gene clustering patterns that can be used as “expression signatures”. The results indicate each toxicant exposure caused between 64 and 222 genes to be significantly altered in expression. Most genes exhibiting altered expression responded to only one of the toxicants and relatively few were co-expressed in multiple treatments. For example, BaP and Diquat, both of which exert toxicity via oxidative stress, upregulated 28 of the same genes, of over 100 genes altered by either treatment. Other genes associated with steroidogenesis, p450 and estrogen responsive genes appear to be useful for selectively identifying toxicant mode of action in fish, suggesting a link between gene expression profile and mode of toxicity. Our array results showed good agreement with quantitative real time polymerase chain reaction (qRT PCR), which demonstrates that the arrays are an accurate measure of gene expression. The specificity of the gene expression profile in response to a model toxicant, the link between genes with altered expression and mode of toxic action, and the consistency between array and qRT PCR results all suggest that cDNA microarrays have the potential to screen environmental contaminants for biomarkers and mode of toxic action. PMID:16488489

Gene expression patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suite of model toxicants.

PubMed

Hook, Sharon E; Skillman, Ann D; Small, Jack A; Schultz, Irvin R

2006-05-25

The increased availability and use of DNA microarrays has allowed the characterization of gene expression patterns associated with exposure to different toxicants. An important question is whether toxicant induced changes in gene expression in fish are sufficiently diverse to allow for identification of specific modes of action and/or specific contaminants. In theory, each class of toxicant may generate a gene expression profile unique to its mode of toxic action. In this study, isogenic (cloned) rainbow trout Oncorhynchus mykiss were exposed to sublethal levels of a series of model toxicants with varying modes of action, including ethynylestradiol (xeno-estrogen), 2,2,4,4'-tetrabromodiphenyl ether (BDE-47, thyroid active), diquat (oxidant stressor), chromium VI, and benzo[a]pyrene (BaP) for a period of 1-3 weeks. An additional experiment measured trenbolone (anabolic steroid; model androgen) induced gene expression changes in sexually mature female trout. Following exposure, fish were euthanized, livers removed and RNA extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNA's. The slides were scanned to measure abundance of a given transcript in each sample relative to controls. Data were analyzed via Genespring (Silicon Genetics) to identify a list of up- and downregulated genes, as well as to determine gene clustering patterns that can be used as "expression signatures". The results indicate each toxicant exposure caused between 64 and 222 genes to be significantly altered in expression. Most genes exhibiting altered expression responded to only one of the toxicants and relatively few were co-expressed in multiple treatments. For example, BaP and Diquat, both of which exert toxicity via oxidative stress, upregulated 28 of the same genes, of over 100 genes altered by either treatment. Other genes associated with steroidogenesis, p450 and estrogen responsive genes appear to be useful for selectively identifying toxicant mode of action in fish, suggesting a link between gene expression profile and mode of toxicity. Our array results showed good agreement with quantitative real time polymerase chain reaction (qRT PCR), which demonstrates that the arrays are an accurate measure of gene expression. The specificity of the gene expression profile in response to a model toxicant, the link between genes with altered expression and mode of toxic action, and the consistency between array and qRT PCR results all suggest that cDNA microarrays have the potential to screen environmental contaminants for biomarkers and mode of toxic action.

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

PubMed

Yamaguchi, Akemi; Matsuda, Kazuyuki; Sueki, Akane; Taira, Chiaki; Uehara, Masayuki; Saito, Yasunori; Honda, Takayuki

2015-08-25

Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

Selection of reference genes for RT-qPCR analysis in tumor tissues from male hepatocellular carcinoma patients with hepatitis B infection and cirrhosis.

PubMed

Liu, Shuang; Zhu, Pengfei; Zhang, Ling; Ding, Shanlong; Zheng, Sujun; Wang, Yang; Lu, Fengmin

2013-01-01

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the high specificity, sensitivity and accuracy of this technique. However, its reliability is strongly depends on the expression stability of reference gene used for data normalization. Therefore, identification of reliable and condition specific reference genes is critical for the success of RT-qPCR. Hepatitis B virus (HBV) infection, male gender and the presence of cirrhosis are widely recognized as the leading independent risk factors for the development of hepatocellular carcinoma (HCC). This study aimed to select reliable reference gene for RT-qPCR analysis in HCC patients with all of those risk factors. Six candidate reference genes were analyzed in 33 paired tumor and non-tumor tissues from untreated HCC patients. The genes expression stabilities were assessed by geNorm and NormFinder. C-terminal binding protein 1(CTBP1) was the most stable gene among the 6 candidate genes evaluated by both geNorm and NormFinder. The expression stability values were 0.08 for CTBP1 and UBC, 0.09 for HPRT1, 0.12 for HMBS, 0.14 for GAPDH and 0.18 for 18S with geNorm analysis. The stability values suggested by NormFinder software were CTBP1: 0.044, UBC: 0.063, HMBS: 0.072, HPRT1: 0.072, GAPDH: 0.098 and 18S rRNA: 0.161. This is the first systematic analysis which suggested CTBP1 as the highest expression-stable gene in human male HBV infection related-HCC with cirrhosis. We recommend CTBP1 as the best candidate reference gene when RT-qPCR was used to determine gene(s) expression in HCC. This may facilitate the relevant HBV related HCC studies in the future.

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

PubMed

Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

2015-11-01

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. Published by Elsevier B.V.

Establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (CPA-LFD) for rapid and specific detection of red-spotted grouper nervous necrosis virus.

PubMed

Su, Zi Dan; Shi, Cheng Yin; Huang, Jie; Shen, Gui Ming; Li, Jin; Wang, Sheng Qiang; Fan, Chao

2015-09-26

Red-spotted grouper nervous necrosis virus (RGNNV) is an important pathogen that causes diseases in many species of fish in marine aquaculture. The larvae and juveniles are more easily infected by RGNNV and the cumulative mortality is as high as 100 % after being infected with RGNNV. This virus imposes a serious threat to aquaculture of grouper fry. This study aimed to establish a simple, accurate and highly sensitive method for rapid detection of RGNNV on the spot. In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification (CPA) system was established. The product amplified by CPA was detected through visualization with lateral flow dipstick (LFD). Three important parameters, including the amplification temperature, the concentration of dNTPs and the concentration of Mg(2+) for the CPA system, were optimized. The sensitivity and specificity of this method for RGNNV were tested and compared with those of the conventional RT-PCR and real-time quantitative RT-PCR (qRT-PCR). The optimized conditions for the CPA amplification system were determined as follows: the optimal amplification temperature, the optimized concentration of dNTPs and the concentration for Mg(2+) were 69 °C, 1.2 mmol/L and 5 mmol/L, respectively. The lowest limit of detection (LLOD) of this method for RGNNV was 10(1) copies/μL of RNA sample, which was 10 times lower than that of conventional RT-PCR and comparable to that of RT-qPCR. This method was specific for RGNNV in combination with SJNNV and had no cross-reactions with 8 types of virus and bacterial strains tested. This method was successfully applied to detect RGNNV in fish samples. This study established a CPA-LFD method for detection of RGNNV. This method is simple and rapid with high sensitivity and good specificity and can be widely applied for rapid detection of this virus on the spot.

Development of mRNA-specific RT-PCR for the detection of koi herpesvirus (KHV) replication stage.

PubMed

Yuasa, Kei; Kurita, Jun; Kawana, Morihiko; Kiryu, Ikunari; Oseko, Norihisa; Sano, Motohiko

2012-08-13

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.

Evaluation of digital PCR for absolute RNA quantification.

PubMed

Sanders, Rebecca; Mason, Deborah J; Foy, Carole A; Huggett, Jim F

2013-01-01

Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

PubMed

Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

2013-09-15

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. Copyright © 2013 Elsevier B.V. All rights reserved.

Selecting a set of housekeeping genes for quantitative real-time PCR in normal and tetraploid haemocytes of soft-shell clams, Mya arenaria.

PubMed

Siah, A; Dohoo, C; McKenna, P; Delaporte, M; Berthe, F C J

2008-09-01

The transcripts involved in the molecular mechanisms of haemic neoplasia in relation to the haemocyte ploidy status of the soft-shell clam, Mya arenaria, have yet to be identified. For this purpose, real-time quantitative RT-PCR constitutes a sensitive and efficient technique, which can help determine the gene expression involved in haemocyte tetraploid status in clams affected by haemic neoplasia. One of the critical steps in comparing transcription profiles is the stability of selected housekeeping genes, as well as an accurate normalization. In this study, we selected five reference genes, S18, L37, EF1, EF2 and actin, generally used as single control genes. Their expression was analyzed by real-time quantitative RT-PCR at different levels of haemocyte ploidy status in order to select the most stable genes. Using the geNorm software, our results showed that L37, EF1 and S18 represent the most stable gene expressions related to various ploidy status ranging from 0 to 78% of tetraploid haemocytes in clams sampled in North River (Prince Edward Island, Canada). However, actin gene expression appeared to be highly regulated. Hence, using it as a housekeeping gene in tetraploid haemocytes can result in inaccurate data. To compare gene expression levels related to haemocyte ploidy status in Mya arenaria, using L37, EF1 and S18 as housekeeping genes for accurate normalization is therefore recommended.

Effects of sanitation, freezing and frozen storage on enteric viruses in berries and herbs.

PubMed

Butot, S; Putallaz, T; Sánchez, G

2008-08-15

Norovirus (NV) and hepatitis A virus (HAV) are foodborne enteric viruses associated with outbreaks of disease following consumption of fresh or frozen produce. Model experiments were performed to determine the effectiveness of certain commercial processes for the removal of enteric viruses that might be present in berries and herbs. The survival and persistence of HAV, NV, rotavirus (RV) and feline calicivirus (FCV), a surrogate for NV, in frozen produce over time were determined. Survival and inactivation of HAV, RV and FCV were assessed by viral culture and quantitative reverse transcription-PCR (RT-PCR), whereas NV persistence was determined by quantitative RT-PCR only. Freezing did not significantly reduce the viability of any of the viruses except the infectivity of FCV in strawberries. Frozen storage for 3 months had limited effects on HAV and RV survival in all tested food products, whereas in frozen raspberries and strawberries FCV infectivity showed the highest decay rate due to acid pH. To simulate postharvesting conditions, fresh berries and herbs were rinsed with tap, warm or chlorinated water or with a chlorine dioxide (ClO(2)) solution. Available chlorine at a concentration of 200 ppm and ClO(2) at 10 ppm reduced measurable enteric viruses in raspberry and parsley samples by less than 2 log(10) units.

Molecular cloning of allelopathy related genes and their relation to HHO in Eupatorium adenophorum.

PubMed

Guo, Huiming; Pei, Xixiang; Wan, Fanghao; Cheng, Hongmei

2011-10-01

In this study, conserved sequence regions of HMGR, DXR, and CHS (encoding 3-hydroxy-3-methylglutaryl-CoA reductase, 1-deoxyxylulose-5-phosphate reductoisomerase and chalcone synthase, respectively) were amplified by reverse transcriptase (RT)-PCR from Eupatorium adenophorum. Quantitative real-time PCR showed that the expression of CHS was related to the level of HHO, an allelochemical isolated from E. adenophorum. Semi-quantitative RT-PCR showed that there was no significant difference in expression of genes among three different tissues, except for CHS. Southern blotting indicated that at least three CHS genes are present in the E. adenophorum genome. A full-length cDNA from CHS genes (named EaCHS1, GenBank ID: FJ913888) was cloned. The 1,455 bp cDNA contained an open reading frame (1,206 bp) encoding a protein of 401 amino acids. Preliminary bioinformatics analysis of EaCHS1 revealed that EaCHS1 was a member of CHS family, the subcellular localization predicted that EaCHS1 was a cytoplasmic protein. To the best of our knowledge, this is the first report of conserved sequences of these genes and of a full-length EaCHS1 gene in E. adenophorum. The results indicated that CHS gene is related to allelopathy of E. adenophorum.

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

PubMed

Yong, Kwon Joong; Milenic, Diane E; Baidoo, Kwamena E; Brechbiel, Martin W

2014-01-01

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

Profiling of the yak skeletal muscle tissue gene expression and comparison with the domestic cattle by genome array.

PubMed

Wang, H B; Zan, L S; Zhang, Y Y

2014-01-01

Of all the mammals of the world, the yak lives at the highest altitude area of more than 3000 m. Comparison between yak and cattle of the low-altitude areas will be informative in studying animal adaptation to higher altitudes. To investigate the molecular mechanism involved in meat quality differences between the two Chinese special varieties Qinghai yak and Qinchuan cattle, 12 chemical-physical characteristics of the longissimus dorsi muscle related to meat quality were compared at the age of 36 months, and the gene expression profiles were constructed by utilizing the bovine genome array. Significant analysis of microarrays was used to identify the differentially expressed genes. Gene ontology and pathway analysis were performed by a free Web-based Molecular Annotation System 2.0. The results reveal ~11 000 probes representing about 10 000 genes that were detected in both the Qinghai yak and Qinchuan cattle. A total of 1922 genes were shown to be differentially expressed, 633 probes were upregulated and 1259 probes were downregulated in the muscle tissue of Qinghai yak that were mainly involved in ubiquitin-mediated proteolysis, muscle growth regulation, glucose metabolism, immune response and so on. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. Further analysis implied that animals living at a high altitude may supply energy by more active protein catabolism and glycolysis compared with those living in the plain areas. Our results establish the groundwork for further studies on yaks' meat quality and will be beneficial in improving the yaks' breeding by molecular biotechnology.

Evidence for changes in the transcription levels of two putative P-glycoprotein genes in sea lice (Lepeophtheirus salmonis) in response to emamectin benzoate exposure.

PubMed

Tribble, Nicholas D; Burka, John F; Kibenge, Frederick S B

2007-05-01

Overexpression of P-glycoproteins (Pgps) is assumed to be a principal mechanism of resistance of nematodes and arthropods to macrocyclic lactones. Quantitative RT-PCR (Q-RT-PCR) was used to demonstrate changes in transcription levels of two putative P-glycoprotein genes, designated here as SL0525 and SL-Pgp1, in sea lice (Lepeophtheirus salmonis) following exposure to emamectin benzoate (EMB). Pre-adult L. salmonis were challenged in an EMB bioassay for 24h and gene expression was studied from lice surviving EMB concentrations of 0, 10, and 30ppb. Gene expression was measured using Q-RT-PCR with elongation factor 1 (eEF1alpha) as an internal reference gene. The results show that both target genes, SL0525 and SL-Pgp1, had significantly increased levels of expression with exposure to 10ppb EMB (p=0.11 and p=0.17, respectively) whereas the group exposed to 30ppb was on the verge of being significant (p=0.053) only in the expression of SL-Pgp1. Gene expression for SL0525 and SL-Pgp1 were increased over five-fold at 10ppb EMB. Therefore, the upregulation of these target genes may offer protection by increasing Pgp expression when lice are exposed to EMB. Our optimized Q-RT-PCR can be used to determine if over-expression of these genes could be the basis for development of resistance in sea lice and thus allow suitable alternative chemotherapeutic options to be assessed.

Biomarkers of the Hedgehog/Smoothened pathway in healthy volunteers

PubMed Central

Kadam, Sunil K; Patel, Bharvin K R; Jones, Emma; Nguyen, Tuan S; Verma, Lalit K; Landschulz, Katherine T; Stepaniants, Sergey; Li, Bin; Brandt, John T; Brail, Leslie H

2012-01-01

The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway. PMID:22611475

Endometrial development and function in experimentally induced luteal phase deficiency.

PubMed

Usadi, Rebecca S; Groll, Jeremy M; Lessey, Bruce A; Lininger, Ruth A; Zaino, Richard J; Fritz, Marc A; Young, Steven L

2008-10-01

It is generally assumed that delayed endometrial development observed in luteal phase deficiency (LPD) is the result of abnormally low progesterone (P) levels. This hypothesis has never been tested by direct experiment. Our objective was to evaluate the effects of P concentrations on human endometrium. A randomized trial was conducted at an academic medical center. Twenty-nine healthy, ovulatory 18- to 35-yr-old women participated. Endometrial samples were obtained from women in natural cycles and two groups of experimentally modeled cycles. Women undergoing modeled cycles were treated with GnRH agonist and a fixed physiological dose of transdermal estradiol, followed by randomization to 10 or 40 mg daily im P administration to achieve either normal circulating luteal P or 4-fold lower P concentrations, the latter representing an experimental model of LPD. Tissue specimens, obtained after 10 days of P exposure, were analyzed by histological dating, immunohistochemistry, immunoblot, and real-time quantitative RT-PCR (qRT-PCR). Histological dating of endometrium, immunohistochemistry for endometrial integrins, and qRT-PCR analysis for nine putative functional markers showed no differences between the three groups. Preliminary data from Western analysis suggest that some proteins may be affected by low serum P concentrations. Histological endometrial dating does not reflect circulating P concentrations and cannot serve as a reliable bioassay of the quality of luteal function. Assessment of selected functional markers by either immunohistochemistry or qRT-PCR is similarly insensitive to decreased circulating P. Preliminary evidence suggests that abnormally low luteal phase serum P concentrations may have important functional consequences not otherwise detected.

Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

PubMed Central

Beld, Marcel; Minnaar, René; Weel, Jan; Sol, Cees; Damen, Marjolein; van der Avoort, Harry; Wertheim-van Dillen, Pauline; Breda, Alex van; Boom, René

2004-01-01

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine α-casein per μl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine α-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens. PMID:15243060

Detection of Helicobacter pylori by Real-Time PCR for 16s rRNA in Stools of NonInfected Healthy Children, Using ELISA Antigen Stool Test as the Gold Standard.

PubMed

George, Sergio; Mamani, Nora; Lucero, Yalda; Torres, Juan Pablo; Farfán, Mauricio; Lagomarcino, Anne J; Orellana, Andrea; O'Ryan, Miguel

2016-12-01

We previously detected Helicobacter pylori infection by stool antigen ELISA assay in 33-41% of asymptomatic Chilean children between 2-3 years of age, of which 11-20% had a transient infection and 21-22% a persistent infection. A total of 88% of ELISA-positive samples were also rtPCR positive, while 37/133 (33%) of ELISA-negative stool samples were rtPCR positive. The significance of a ELISA-negative/rtPCR-positive sample requires clarification. We aimed to determine whether rtPCR is able to detect persistent infections not detected by ELISA. We selected 36 children with an ELISA-negative/rtPCR-positive stool sample, of which 25 were never H. pylori infected according to ELISA, and 11 had a transient infection with an ELISA-positive sample before or after the discordant sample. At least two additional consecutive ELISA-negative samples per child were tested in duplicate by rtPCR for the 16s rRNA gene. A total of 14 of 78 (17.9%) rtPCR reactions were positive, but only 4/78 (5.1%) were positive in both duplicates, representing a total of 3/36 (8.3%) children with an additional rtPCR-positive sample, only one of whom was persistently negative by ELISA. One child with a transient infection had two positive rtPCR reactions despite negative ELISA samples. In H. pylori noninfected or transiently infected children, as determined by stool ELISA, additional ELISA-negative/rtPCR-positive stool samples were found in 8.3% of children, but a possible persistent infection was only identified in 2.7% of children. Thus, the characterization of infection dynamics in children is not being misrepresented by application of stool ELISA. Furthermore, rtPCR does not significantly improve dynamic characterization. © 2016 John Wiley & Sons Ltd.

EPConDB: a web resource for gene expression related to pancreatic development, beta-cell function and diabetes.

PubMed

Mazzarelli, Joan M; Brestelli, John; Gorski, Regina K; Liu, Junmin; Manduchi, Elisabetta; Pinney, Deborah F; Schug, Jonathan; White, Peter; Kaestner, Klaus H; Stoeckert, Christian J

2007-01-01

EPConDB (http://www.cbil.upenn.edu/EPConDB) is a public web site that supports research in diabetes, pancreatic development and beta-cell function by providing information about genes expressed in cells of the pancreas. EPConDB displays expression profiles for individual genes and information about transcripts, promoter elements and transcription factor binding sites. Gene expression results are obtained from studies examining tissue expression, pancreatic development and growth, differentiation of insulin-producing cells, islet or beta-cell injury, and genetic models of impaired beta-cell function. The expression datasets are derived using different microarray platforms, including the BCBC PancChips and Affymetrix gene expression arrays. Other datasets include semi-quantitative RT-PCR and MPSS expression studies. For selected microarray studies, lists of differentially expressed genes, derived from PaGE analysis, are displayed on the site. EPConDB provides database queries and tools to examine the relationship between a gene, its transcriptional regulation, protein function and expression in pancreatic tissues.

Chronic myelogenous leukemia: laboratory diagnosis and monitoring.

PubMed

Wang, Y L; Bagg, A; Pear, W; Nowell, P C; Hess, J L

2001-10-01

Rapid developments have occurred both in laboratory medicine and in therapeutic interventions for the management of patients with chronic myelogenous leukemia (CML). With a wide array of laboratory tests available, selecting the appropriate test for a specific diagnostic or therapeutic setting has become increasingly difficult. In this review, we first discuss, from the point of view of laboratory medicine, the advantages and disadvantages of several commonly used laboratory assays, including cytogenetics, fluorescence in situ hybridization (FISH), and qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We then discuss, from the point of view of clinical care, the test(s) of choice for the most common clinical scenarios, including diagnosis and monitoring of the therapeutic response and minimal residual disease in patients treated with different therapies. The purpose of this review is to help clinicians and laboratory physicians select appropriate tests for the diagnosis and monitoring of CML, with the ultimate goal of improving the cost-effective usage of clinical laboratories and improving patient care. Copyright 2001 Wiley-Liss, Inc.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

PubMed

Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le

2017-11-01

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.

Malaria prevalence in asymptomatic and symptomatic children in Kiwangwa, Bagamoyo district, Tanzania.

PubMed

Sumari, Deborah; Mwingira, Felista; Selemani, Majige; Mugasa, Joseph; Mugittu, Kefas; Gwakisa, Paul

2017-05-25

Malaria prevalence continues to decline across sub-Saharan Africa as a result of various intervention strategies. However, the diseases still poses a public health concern in the region. While symptomatic malaria is recognized and treated, asymptomatic infections become increasingly important for interrupting transmission. A cross-sectional survey was conducted to assess malaria prevalence in symptomatic and asymptomatic children in Kiwangwa ward in Bagamoyo District in Tanzania. Four hundred school-aged children in Kiwanga ward were recruited in the study; 200 from Kiwangwa dispensary and 200 from nearby schools. Primary health parameters were examined and blood samples collected and examined for Plasmodium falciparum prevalence using rapid diagnostic test (RDT), light microscopy (LM) and reverse transcription quantitative PCR (RT-qPCR) targeting transcripts of A-type 18s rRNA of P. falciparum. Gametocytes were detected by LM and RT-qPCR targeting transcripts of gametocyte specific marker, Pfs25. Overall P. falciparum prevalence was 73.3, 40.8 and 36.3% by RT-qPCR, RDT and LM in the study area, respectively (P < 0.001). As expected symptomatic children had a significantly higher prevalence of 89, 67.5 and 64.5% by qPCR, RDT and LM, compared to 57.5, 14 and 8% in the asymptomatic group, respectively. However, gametocyte prevalence in asymptomatic individuals was higher by both LM (2%) and qPCR (14%) than in symptomatic individuals LM (0.5%) and qPCR (3%). A substantial difference in prevalence of symptomatic and asymptomatic infections observed in Kiwangwa ward underpins the use of molecular tools in malaria surveillance aiming at estimating prevalence and transmission. Notably, the higher gametocytaemia observed in asymptomatic children indicates the reservoir infections and points to the need for detection and treatment of both asymptomatic and symptomatic malaria.

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

PubMed Central

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes. PMID:25793735

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

PubMed

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa).

PubMed

Song, Hao; Dang, Xin; He, Yuan-Qiu; Zhang, Tao; Wang, Hai-Yan

2017-01-01

The veined rapa whelk Rapana venosa is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in R. venosa . For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in R. venosa for use as internal controls for qRT-PCR. In this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1 α ( EF-1α ), α -actin ( ACT ), cytochrome c oxidase subunit 1 ( COX1 ), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 α subcomplex subunit 7 ( NDUFA7 ), 60S ribosomal protein L5 ( RL5 ), 60S ribosomal protein L28 ( RL28 ), glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ), β -tubulin ( TUBB ), 40S ribosomal protein S25 ( RS25 ), 40S ribosomal protein S8 ( RS8 ), ubiquitin-conjugating enzyme E2 ( UBE2 ), histone H3 ( HH3 ), and peptidyl-prolyl cis-trans isomerase A ( PPIA ). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms. Of the 13 candidate genes tested, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. For the normalization of a single specific tissue, we suggested that EF-1α and NDUFA7 are the best combination in gonad, as well as COX1 and RL28 for intestine, EF-1α and RL5 for kidney, EF-1α and COX1 for gill, EF-1α and RL28 for Leiblein and mantle, EF-1α , RL5 , and NDUFA7 for liver , GAPDH , PPIA , and RL28 for hemocyte. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, PPIA , RS25 , and RL28 for stage 1, RL5 and RL28 for stage 2 and 5, RL28 and NDUFA7 for stage 3, and PPIA and TUBB for stage 4. Our results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of R. venosa in the future.

Functional analyses of the digestive ß-Glucosidase of Formosan Subterranean Termites (Coptotermes formosanus)

USDA-ARS?s Scientific Manuscript database

The research was to elucidate the function of the ß-glucosidase of Formosan subterranean termites in vitro and in vivo. Quantitative RT-PCR analyses indicated that the gene transcript was relatively more abundant in the foraging worker caste than in other castes and salivary glands were the major ex...

Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.

PubMed

Butler, Alexandra E; Matveyenko, Aleksey V; Kirakossian, David; Park, Johanna; Gurlo, Tatyana; Butler, Peter C

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6-7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

A quantitative real-time RT-PCR assay to measure TGF-beta mRNA and its correlation with hematologic, plasma chemistry and organo-somatic indices responses in triamcinolone-treated Atlantic menhaden, Brevoortia tyrannus.

PubMed

Johnson, A K; Harms, C A; Levine, J F; Law, J McHugh

2006-01-01

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Surveillance for Western equine encephalitis St. Louis encephalitis and West Nile viruses using reverse transcription loop-mediated isothermal amplification

DOE PAGES

Meagher, Robert J.; Ball, Cameron Scott; Langevin, Stanley A.; ...

2016-01-25

In this study, collection of mosquitoes and testing for vector-borne viruses is a key surveillance activity that directly influences the vector control efforts of public health agencies, including determining when and where to apply insecticides. Vector control districts in California routinely monitor for three human pathogenic viruses including West Nile virus (WNV), Western equine encephalitis virus (WEEV), and St. Louis encephalitis virus (SLEV). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) offers highly sensitive and specific detection of these three viruses in a single multiplex reaction, but this technique requires costly, specialized equipment that is generally only available in centralized publicmore » health laboratories. We report the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect WNV, WEEV, and SLEV RNA extracted from pooled mosquito samples collected in California, including novel primer sets for specific detection of WEEV and SLEV, targeting the nonstructural protein 4 (nsP4) gene of WEEV and the 3’ untranslated region (3’-UTR) of SLEV. Our WEEV and SLEV RT-LAMP primers allowed detection of <0.1 PFU/reaction of their respective targets in <30 minutes, and exhibited high specificity without cross reactivity when tested against a panel of alphaviruses and flaviviruses. Furthermore, the SLEV primers do not cross-react with WNV, despite both viruses being closely related members of the Japanese encephalitis virus complex. The SLEV and WEEV primers can also be combined in a single RT-LAMP reaction, with discrimination between amplicons by melt curve analysis. Although RT-qPCR is approximately one order of magnitude more sensitive than RT-LAMP for all three targets, the RT-LAMP technique is less instrumentally intensive than RT-qPCR and provides a more cost-effective method of vector-borne virus surveillance.« less

Immunocapture reverse transcription-polymerase chain reaction combined with nested PCR greatly increases the detection of Prunus necrotic ring spot virus in the peach.

PubMed

Helguera, P R; Taborda, R; Docampo, D M; Ducasse, D A

2001-06-01

A detection system based on nested PCR after IC-RT-PCR (IC-RT-PCR-Nested PCR) was developed to improve indexing of Prunus necrotic ringspot virus in peach trees. Inhibitory effects and inconsistencies of the standard IC-RT-PCR were overcome by this approach. IC-RT-PCR-Nested PCR improved detection by three orders of magnitude compared with DAS-ELISA for the detection of PNRSV in leaves. Several different tissues were evaluated and equally consistent results were observed. The main advantages of the method are its consistency, high sensitivity and easy application in quarantine programs.

Evaluation of a sensitive reverse transcription PCR-enzymelinked immunosorbent assay for detection of hepatitis A virus in oysters (Saccostrea glomerata) on the east coast of the Gulf of Thailand.

PubMed

Intamaso, Uraiwan; Ketkhunthod, Sitthisak

2014-05-01

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

Comparison of 2 Assays for Diagnosing Rotavirus and Evaluating Vaccine Effectiveness in Children with Gastroenteritis

PubMed Central

Mijatovic-Rustempasic, Slavica; Tam, Ka Ian; Lyde, Freda C.; Payne, Daniel C.; Szilagyi, Peter; Edwards, Kathryn; Staat, Mary Allen; Weinberg, Geoffrey A.; Hall, Caroline B.; Chappell, James; McNeal, Monica; Gentsch, Jon R.; Bowen, Michael D.; Parashar, Umesh D.

2013-01-01

We compared rotavirus detection rates in children with acute gastroenteritis (AGE) and in healthy controls using enzyme immunoassays (EIAs) and semiquantitative real-time reverse transcription PCR (qRT-PCR). We calculated rotavirus vaccine effectiveness using different laboratory-based case definitions to determine which best identified the proportion of disease that was vaccine preventable. Of 648 AGE patients, 158 (24%) were EIA positive, and 157 were also qRT-PCR positive. An additional 65 (10%) were qRT-PCR positive but EIA negative. Of 500 healthy controls, 1 was EIA positive and 24 (5%) were qRT-PCR positive. Rotavirus vaccine was highly effective (84% [95% CI 71%–91%]) in EIA-positive children but offered no significant protection (14% [95% CI −105% to 64%]) in EIA-negative children for whom virus was detected by qRT-PCR alone. Children with rotavirus detected by qRT-PCR but not by EIA were not protected by vaccination, suggesting that rotavirus detected by qRT-PCR alone might not be causally associated with AGE in all patients. PMID:23876518

Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

PubMed

Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

2018-06-01

We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma.

PubMed

Gai, Chiara; Camussi, Francesco; Broccoletti, Roberto; Gambino, Alessio; Cabras, Marco; Molinaro, Luca; Carossa, Stefano; Camussi, Giovanni; Arduino, Paolo G

2018-04-18

Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs. OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves. A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC. In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

Identification of innovative potential quality markers in rocket and melon fresh-cut produce.

PubMed

Cavaiuolo, Marina; Cocetta, Giacomo; Bulgari, Roberta; Spinardi, Anna; Ferrante, Antonio

2015-12-01

Ready-to-eat fresh cut produce are exposed to pre- and postharvest abiotic stresses during the production chain. Our work aimed to identify stress responsive genes as new molecular markers of quality that can be widely applied to leaves and fruits and easily determined at any stage of the production chain. Stress responsive genes associated with quality losses were isolated in rocket and melon fresh-cut produce and their expression levels analyzed by quantitative real time PCR (qRT-PCR) at different time points after harvest at 20 °C and 4 °C. qRT-PCR results were supported by correlation analysis with physiological and biochemical determinations evaluated at the same conditions such as chlorophyll a fluorescence indices, total, reducing sugars, sucrose, ethylene, ascorbic acid, lipid peroxidation and reactive oxygen species. In both species the putative molecular markers increased their expression soon after harvest suggesting a possible use as novel and objective quality markers of fresh-cut produces. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PubMed Central

Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

2014-01-01

Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs. PMID:24752452

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

PubMed Central

Ma, Rui; Xu, Sheng; Zhao, Yucheng; Xia, Bing; Wang, Ren

2016-01-01

Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions. PMID:27200013

Comparative evaluation of fluorescent in situ hybridization and Giemsa microscopy with quantitative real-time PCR technique in detecting malaria parasites in a holoendemic region of Kenya.

PubMed

Osoga, Joseph; Waitumbi, John; Guyah, Bernard; Sande, James; Arima, Cornel; Ayaya, Michael; Moseti, Caroline; Morang'a, Collins; Wahome, Martin; Achilla, Rachel; Awinda, George; Nyakoe, Nancy; Wanja, Elizabeth

2017-07-24

Early and accurate diagnosis of malaria is important in treatment as well as in the clinical evaluation of drugs and vaccines. Evaluation of Giemsa-stained smears remains the gold standard for malaria diagnosis, although diagnostic errors and potential bias estimates of protective efficacy have been reported in practice. Plasmodium genus fluorescent in situ hybridization (P-Genus FISH) is a microscopy-based method that uses fluorescent labelled oligonucleotide probes targeted to pathogen specific ribosomal RNA fragments to detect malaria parasites in whole blood. This study sought to evaluate the diagnostic performance of P-Genus FISH alongside Giemsa microscopy compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR) in a clinical setting. Five hundred study participants were recruited prospectively and screened for Plasmodium parasites by P-Genus FISH assay, and Giemsa microscopy. The microscopic methods were performed by two trained personnel and were blinded, and if the results were discordant a third reading was performed as a tie breaker. The diagnostic performance of both methods was evaluated against qRT-PCR as a more sensitive method. The number of Plasmodium positive cases was 26.8% by P-Genus FISH, 33.2% by Giemsa microscopy, and 51.2% by qRT-PCR. The three methods had 46.8% concordant results with 61 positive cases and 173 negative cases. Compared to qRT-PCR the sensitivity and specificity of P-Genus FISH assay was 29.3 and 75.8%, respectively, while microscopy had 58.2 and 93.0% respectively. Microscopy had a higher positive and negative predictive values (89.8 and 68.0% respectively) compared to P-Genus FISH (56.0 and 50.5%). In overall, microscopy had a good measure of agreement (76%, k = 0.51) compared to P-Genus FISH (52%, k = 0.05). The diagnostic performance of P-Genus FISH was shown to be inferior to Giemsa microscopy in the clinical samples. This hinders the possible application of the method in the field despite the many advantages of the method especially diagnosis of low parasite density infections. The P-Genus assay has great potential but application of the method in clinical setting would rely on extensive training of microscopist and continuous proficiency testing.

Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

PubMed

Xu, Chun-Wei; Wang, Wen-Xian; Chen, Yan-Ping; Chen, Yu; Liu, Wei; Zhong, Li-Hua; Chen, Fang-Fang; Zhuang, Wu; Song, Zheng-Bo; Chen, Xiao-Hui; Huang, Yun-Jian; Guan, Yan-Fang; Yi, Xin; Lv, Tang-Feng; Zhu, Wei-Feng; Lu, Jian-Ping; Wang, Xiao-Jiang; Shi, Yi; Lin, Xian-Dong; Chen, Gang; Song, Yong

2018-04-11

ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for ALK-targeted therapy. VENTANA IHC has moderate sensitivity and a slightly higher association with response to therapy with ALK inhibitors, and some VENTANA IHC-positive, but RT-PCR-negative cases may benefit from crizotinib.

Global microRNA profiling of peripheral blood mononuclear cells in patients with Behçet's disease.

PubMed

Erre, Gian Luca; Piga, Matteo; Carru, Ciriaco; Angius, Andrea; Carcangiu, Laura; Piras, Marco; Sotgia, Salvatore; Zinellu, Angelo; Mathieu, Alessandro; Passiu, Giuseppe; Pescatori, Mario

2015-01-01

To explore the post-transcriptional regulation of the peripheral blood mononuclear cells (PBMCs) transcriptome by microRNAs in Behçet's disease (BD). Using TaqMan Low Density Array-based microRNAs expression profiling, the expression of 750 mature human microRNAs in PBMCs from 5 BD patients and 3 healthy controls (HC) was compared. The expression of deregulated microRNAs was then validated by quantitative real time-polymerase chain reaction (qRT-PCR), in 42 BD patients and 8 HC. In the initial screening, 13 microRNAs appeared deregulated in BD vs HC. Among them, the differential expression of miR-720 and miR-139-3p was confirmed by qRT-PCR, (p<0.05 and FDR<5%). Areas under the receiver operating characteristic curve for miR-139-3p, miR-720 and miR-139-3p+miR-720 in the validation cohort were 0.84, 0.87 and 0.92 respectively, indicating good discrimination between BD patients and HC. Post-hoc analysis showed that 9 out of 13 microRNAs from the discovery phase were significantly upregulated in active vs. quiescent BD, suggesting inflammation as a key regulator of microRNAs machinery in BD. In silico analysis revealed that several BD candidate susceptibility genes are predicted target of significantly deregulated microRNAs in active BD. A significant enrichment in microRNAs targeting elements of the Toll-like receptor (TLR) and T-cell receptor signalling pathways was also assumed. miR199-3p and miR720 deserve further confirmation as biomarkers of BD in larger studies. PBMCs from active BD displayed a unique signature of microRNAs which may be implicated in regulation of innate immunity activation and T-cell function.

Expression of a hindlimb-determining factor Pitx1 in the forelimb of the lizard Pogona vitticeps during morphogenesis.

PubMed

Melville, Jane; Hunjan, Sumitha; McLean, Felicity; Mantziou, Georgia; Boysen, Katja; Parry, Laura J

2016-10-01

With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the central bearded dragon (Pogona vitticeps) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (GHR, Pitx1 and Shh) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimbs and hindlimbs of P. vitticeps. While the highest level of GHR expression occurred at the hatchling stage, Pitx1 and Shh expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of Pitx1 expression-a hindlimb-determining gene-in the forelimbs of P. vitticeps to that in a closely related Australian agamid lizard, Ctenophorus pictus, where we found Pitx1 expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis-a result consistent with that found across other tetrapods. Expression of Pitx1 in forelimbs has only rarely been documented, including via in situ hybridization in a chicken and a frog. Our findings from both RT-qPCR and IHC indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology. © 2016 The Authors.

Concentration dependent transcriptome responses of zebrafish embryos after exposure to cadmium, cobalt and copper.

PubMed

Sonnack, Laura; Klawonn, Thorsten; Kriehuber, Ralf; Hollert, Henner; Schäfers, Christoph; Fenske, Martina

2017-12-01

Environmental metals are known to cause harmful effects to fish of which many molecular mechanisms still require elucidation. Particularly concentration dependence of gene expression effects is unclear. Focusing on this matter, zebrafish embryo toxicity tests were used in combination with transcriptomics. Embryos were exposed to three concentrations of copper (CuSO 4 ), cadmium (CdCl 2 ) and cobalt (CoSO 4 ) from just after fertilization until the end of the 48hpf pre- and 96hpf post-hatch stage. The RNA was then analyzed on Agilent's Zebrafish (V3, 4×44K) arrays. Enrichment for GO terms of biological processes illustrated for cadmium that most affected GO terms were represented in all three concentrations, while for cobalt and copper most GO terms were represented in the lowest test concentration only. This suggested a different response to the non-essential cadmium than cobalt and copper. In cobalt and copper treated embryos, many developmental and cellular processes as well as the Wnt and Notch signaling pathways, were found significantly enriched. Also, different exposure concentrations affected varied functional networks. In contrast, the largest clusters of enriched GO terms for all three concentrations of cadmium included responses to cadmium ion, metal ion, xenobiotic stimulus, stress and chemicals. However, concentration dependence of mRNA levels was evident for several genes in all metal exposures. Some of these genes may be indicative of the mechanisms of action of the individual metals in zebrafish embryos. Real-time quantitative RT-PCR (qRT-PCR) verified the microarray data for mmp9, mt2, cldnb and nkx2.2a. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel Chromosomal Rearrangements and breakpoints at the t(6;9) in Salivary Adenoid Cystic Carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome

PubMed Central

Mitani, Yoshitsugu; Rao, Pulivarthi H.; Futreal, P. Andrew; Roberts, Dianna B.; Stephens, Philip J.; Zhao, Yi-Jue; Zhang, Li; Mitani, Mutsumi; Weber, Randal S.; Lippman, Scott M.; Caulin, Carlos; El-Naggar, Adel K.

2011-01-01

Objective To investigate the molecular-genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome. Experimental Design Multi-molecular and genetic techniques complemented with massive pair-ended sequencing and SNP array analyses were used on tumor specimens from 30 new and 52 previously RT-PCR analyzed fusion transcript negative ACCs. MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients’ survival. Results The FISH analysis showed 34/82 (41.5%) fusion positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% while fusion transcript forming incidence was 38.2%. Significant statistical association between the 5′ MYB transcript expression and patient survival was found. Conclusions We conclude that: 1) t(6;9) results in a complex genetic and molecular alterations in ACC, 2) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, 3) non-canonical MYB, NFIB gene fusions occur in a subset of tumors, 4) high MYB expression correlates with worse patient survival. PMID:21976542

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

PubMed

Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

2012-11-02

HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

PubMed Central

Masvidal, Laia; Igreja, Susana; Ramos, Maria D; Alvarez, Antoni; de Gracia, Javier; Ramalho, Anabela; Amaral, Margarida D; Larriba, Sara; Casals, Teresa

2014-01-01

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression. PMID:24129438

[Phenotypic and genetic analysis of a patient presented with Tietz/Waardenburg type II a syndrome].

PubMed

Wang, Huanhuan; Tang, Lifang; Zhang, Jingmin; Hu, Qin; Chen, Yingwei; Xiao, Bing

2015-08-01

To determine the genetic cause for a patient featuring decreased pigmentation of the skin and iris, hearing loss and multiple congenital anomalies. Routine chromosomal banding was performed to analyze the karyotype of the patient and his parents. Single nucleotide polymorphism array (SNP array) was employed to identify cryptic chromosome aberrations, and quantitative real-time PCR was used to confirm the results. Karyotype analysis has revealed no obvious anomaly for the patient and his parents. SNP array analysis of the patient has demonstrated a 3.9 Mb deletion encompassing 3p13p14.1, which caused loss of entire MITF gene. The deletion was confirmed by quantitative real-time PCR. Clinical features of the patient have included severe bilateral hearing loss, decreased pigmentation of the skin and iris and multiple congenital anomalies. The patient, carrying a 3p13p14.1 deletion, has features of Tietz syndrome/Waardenburg syndrome type IIa. This case may provide additional data for the study of genotype-phenotype correlation of this disease.

Enterovirus D68 detection in respiratory specimens: Association with severe disease.

PubMed

Engelmann, Ilka; Fatoux, Marie; Lazrek, Mouna; Alidjinou, Enagnon K; Mirand, Audrey; Henquell, Cécile; Dewilde, Anny; Hober, Didier

2017-07-01

Molecular techniques increased the number of documented respiratory infections. In a substantial number of cases the causative agent remains undetected. Since August 2014, an increase in Enterovirus(EV)-D68 infections was reported. We aimed to investigate epidemiology and clinical relevance of EV-D68. From June to December 2014 and from September to December 2015, 803 and 847 respiratory specimens, respectively, were tested for respiratory viruses with a multiplex RT-PCR. This multiplex RT-PCR does not detect EV-D68. Therefore, 457 (2014) and 343 (2015) specimens with negative results were submitted to an EV-specific-RT-PCR. EV-positive specimens were tested with an EV-D68-specific-RT-PCR and genotyped. Eleven specimens of 2014 tested positive in the EV-specific-RT-PCR and of these seven were positive in the EV-D68-specific-RT-PCR. Typing confirmed these as EV-D68. Median age of EV-D68-positive patients was 3 years (1 month-91 years). Common symptoms included fever (n = 6, 86%), respiratory distress (n = 5, 71%), and cough (n = 4, 57%). All EV-D68-positive patients were admitted to hospital, 4 (57%) were admitted to intensive care units and 6 (86%) received oxygen. One patient suffered from acute flaccid paralysis. Seven specimens of 2015 were positive in the EV-specific-RT-PCR but negative in the EV-D68-specific-RT-PCR. In conclusion, use of an EV-specific-RT-PCR allowed us to detect EV-D68 circulation in autumn 2014 that was not detected by the multiplex RT-PCR and was associated with severe disease. © 2017 Wiley Periodicals, Inc.

Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus.

PubMed

Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun

2011-07-01

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Performance of nested RT-PCR on CSF for tuberculous meningitis diagnosis in HIV-infected patients.

PubMed

Gualberto, F A S; Gonçalves, M G; Fukasawa, L O; Santos, A M Ramos Dos; Sacchi, C T; Harrison, L H; Boulware, D R; Vidal, J E

2017-10-01

Timely diagnosis of tuberculous meningitis (TBM) in patients with human immunodeficiency virus (HIV) infection remains a challenge. Despite the current scale-up of the Xpert® MTB/RIF assay, other molecular diagnostic tools are necessary, particularly in referral centres in low- and middle-income countries without Xpert testing. To determine the diagnostic performance of nested real-time polymerase chain reaction (nRT-PCR) in HIV-infected TBM patients categorised according to standardised clinical case definitions. Based on clinical, laboratory and imaging data, HIV-infected patients with suspected TBM were prospectively categorised as 'definite TBM', 'probable TBM', 'possible TBM' or 'not TBM'. We evaluated nRT-PCR sensitivity and specificity in diagnosing TBM among definite TBM cases, and among definite + probable TBM cases. Ninety-two participants were enrolled in the study. nRT-PCR sensitivity for definite TBM (n = 8) was 100% (95%CI 67-100) and 86% (95%CI 60-96) for both definite and probable TBM (n = 6). Assuming that 'not TBM' patients (n = 74) were true-negatives, nRT-PCR specificity was 100% (95%CI 95-100). The possible TBM group (n = 4) had no nRT-PCR positives. The nRT-PCR is a useful rule-in test for HIV-infected patients with TBM according to international consensus case definitions. As nRT-PCR cannot exclude TBM, studies comparing and combining nRT-PCR with other assays are necessary for a rule-out test.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

PubMed

Nishiura, T; Abe, K

1999-01-01

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

[Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].

PubMed

Jin, Mengtong; Sun, Wenshuo; Li, Qin; Sun, Xiaohong; Pan, Yingjie; Zhao, Yong

2014-04-04

We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR). The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences. The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%. Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

Gene expression profiling in melasma in Korean women.

PubMed

Chung, Bo Young; Noh, Tai Kyung; Yang, Sang Hwa; Kim, Il Hwan; Lee, Mi Woo; Yoon, Tae Jin; Chang, Sung Eun

2014-01-01

There has been a paucity of data about the difference in gene expression between melasma lesional skin and normal adjacent one. Our aim was to identify novel genes involved in the pathogenesis of melasma. We performed a microarray analysis and confirmed the results on quantitative real-time polymerase chain reaction (qRT-PCR) in Korean women with melasma. There were 334 genes whose degree of expression showed a significant difference between melasma lesional skin and normal adjacent one. Of these, five were confirmed on qRT-PCR. In melasma lesional skin, there were down-regulation of genes involved in the PPAR signaling pathway and up-regulation of genes involved in neuronal component and the functions of stratum corneum barrier. This result suggests that the pathogenesis of melasma might be associated with novel genes involved in the above signaling pathway in Korean women.

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

PubMed Central

2014-01-01

Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual. PMID:24884716