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Sample records for rna isolation method

  1. miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods.

    PubMed

    Nagy, Zsófia Brigitta; Wichmann, Barnabás; Kalmár, Alexandra; Barták, Barbara Kinga; Tulassay, Zsolt; Molnár, Béla

    2016-07-01

    MiRNA remain stable for detection and PCR-based amplification in FFPE tissue samples. Several miRNA extraction kits are available, however miRNA fraction, as part of total RNA can be isolated using total RNA purification methods, as well. Our primary aim was to compare four different miRNA and total RNA isolation methods from FFPE tissues. Further purposes were to evaluate quantitatively and qualitatively the yield of the isolated miRNA. MiRNAs were isolated from normal colorectal cancer FFPE specimens from the same patients. Two miRNA isolation kits (High Pure miRNA Isolation Kit, miRCURY™ RNA Isolation Kit) and two total RNA isolation kits were compared (High Pure RNA Paraffin Kit, MagNA Pure 96 Cellular RNA LV Kit). Quantity and quality were determined, expression analysis was performed by real-time PCR using qPCR Human Panel I + II (Exiqon) method detecting 742 human miRNAs in parallel. The yield of total RNA was found to be higher than miRNA purification protocols (in CRC: Ex: 0203 ± 0021 μg; HPm: 1,45 ± 0,8 μg; HPp: 21,36 ± 4,98 μg; MP: 8,6 ± 5,1 μg). MiRNAs were detected in lower relative quantity of total RNA compared to the miRNA kits. Higher number of miRNAs could be detected by the miRNA isolation kits in comparison to the total RNA isolation methods. (Ex: 497 ± 16; HPm: 542 ± 11; HPp: 332 ± 36; MP: 295 ± 74). Colon specific miRNAs (miR-21-5p;-34-5p) give satisfying results by miRNA isolation kits. Although miRNA can be detected also after total RNA isolation methods, for reliable and reproducible miRNA expression profiling the use of miRNA isolation kits are more suitable. PMID:26678076

  2. A microRNA isolation method from clinical samples

    PubMed Central

    Zununi Vahed, Sepideh; Barzegari, Abolfazl; Rahbar Saadat, Yalda; Mohammadi, Somayeh; Samadi, Nasser

    2016-01-01

    Introduction: microRNAs (miRNAs) are considered to be novel molecular biomakers that could be exploited in the diagnosis and treatment of different diseases. The present study aimed to develop an efficient miRNA isolation method from different clinical specimens. Methods: Total RNAs were isolated by Trizol reagent followed by precipitation of the large RNAs with potassium acetate (KCH3COOH), polyethylene glycol (PEG) 4000 and 6000, and lithium chloride (LiCl). Then, small RNAs were enriched and recovered from the supernatants by applying a combination of LiCl and ethanol. The efficiency of the method was evaluated through the quality, quantity, and integrity of the recovered RNAs using the A260/280 absorbance ratio, reverse transcription PCR (RT-PCR), and quantitative real-time PCR (q-PCR). Results: Comparison of different RNA isolation methods based on the precipitation of DNA and large RNAs, high miRNA recovery and PCR efficiency revealed that applying potassium acetate with final precipitation of small RNAs using 2.5 M LiCl plus ethanol can provide high yield and quality small RNAs that can be exploited for clinical purposes. Conclusion: The current isolation method can be applied for most clinical samples including cells, formalin-fixed and paraffin-embedded (FFPE) tissues and even body fluids with a wide applicability in molecular biology investigations. PMID:27340621

  3. A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba

    PubMed Central

    Ouyang, Kunxi; Li, Juncheng; Huang, Hao; Que, Qingmin; Li, Pei; Chen, Xiaoyang

    2014-01-01

    Plant tissues contain abundant polysaccharides, phenolic compounds and other metabolites, which makes it difficult to isolate high-quality RNA from them. In addition, Neolamarckia cadamba contains large quantities of other components, particularly RNA-binding alkaloids, which makes the isolation even more challenging. Here, we describe a concise and efficient RNA isolation method that combines the cetyltrimethyl ammonium bromide (CTAB) and Plant RNA Kit (Omega) protocols. Gel electrophoresis showed that RNA extracted from all tissues, using this protocol, was of good integrity and without DNA contamination. Furthermore, the isolated RNA was of high purity, with an A 260/A 280 ratio of 2.1 and an A 260/A 230 ratio of >2.0. The isolated RNA was also suitable for downstream applications, such as reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). The RNA isolation method was also efficient for recalcitrant plant tissues. PMID:26019587

  4. Evaluation of isolation methods for bacterial RNA quantitation in Dickeya dadantii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Modified method for combined DNA and RNA isolation from peanut and other oil seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA...

  6. Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction.

    PubMed

    Tanriverdi, Kahraman; Kucukural, Alper; Mikhalev, Ekaterina; Tanriverdi, Selim E; Lee, Rosalind; Ambros, Victor R; Freedman, Jane E

    2016-05-15

    MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)-qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids. PMID:26969789

  7. An effective method of RNA isolation from Fallopia multiflora tuberous roots.

    PubMed

    Chen, Lei; Sheng, Shu-Jing; Tan, Xue-Mei; Shen, Yan-Jing; Li, Hong-Qing; Zhao, Shu-Jin

    2012-01-01

    To isolate high-quality total RNA from Fallopia multiflora tuberous roots is difficult because of the presence of high levels of carbohydrates, phenolics, and other secondary metabolites. Since several procedures specialized for RNA isolation from polysaccharides and phenols rich tissues have resulted in poor yields, in this study, we developed a modified protocol that was derived from the traditional CTAB method. The protocol was able to produce high-quality and intact RNA from the tuberous roots of F. multiflora. The yield of total RNA was more than 0.15 mg/g fresh weight, with an A260/A280 ratio of 1.9-2.0. The obtained RNA was of sufficient quality and suitable for downstream application such as reverse-transcription polymerase chain reaction (RT-PCR), Northern hybridization, and cDNA library construction. The protocol may also have wider applicability for total RNA isolation from other plant species with tuberous roots. PMID:22239710

  8. An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

    PubMed Central

    Lorenz, W. Walter; Yu, Yuan-Sheng; Dean, Jeffrey F. D.

    2010-01-01

    are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations. PMID:20177393

  9. Comparative studies of two methods for miRNA isolation from milk whey*

    PubMed Central

    Jin, Xiao-lu; Wei, Zi-hai; Liu, Lan; Liu, Hong-yun; Liu, Jian-xin

    2015-01-01

    MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS® followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS® followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (<200 nt) from milk whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100). PMID:26055915

  10. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

    PubMed Central

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

    2016-01-01

    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or

  11. Comparative studies of two methods for miRNA isolation from milk whey.

    PubMed

    Jin, Xiao-lu; Wei, Zi-hai; Liu, Lan; Liu, Hong-yun; Liu, Jian-xin

    2015-06-01

    MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS(®) followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS(®) followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (<200 nt) from milk whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100). PMID:26055915

  12. tRid, an enabling method to isolate previously inaccessible small RNA fractions.

    PubMed

    Futai, Kazuki; Terasaka, Naohiro; Katoh, Takayuki; Suga, Hiroaki

    2016-08-15

    Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coli. PMID:27163863

  13. Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

    SciTech Connect

    Piao, Hailan; Markillie, Lye Meng; Culley, David E.; Mackie, Roderick I.; Hess, Matthias

    2013-03-28

    Metatranscriptomics—gene express profiling via DNA sequencing—is a powerful tool to identify genes that are ac- tively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial commu- nity (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhouse gas twenty times more potent than carbon dioxide, and is of general importance for the physio- logical wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socio- economically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accu- rate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-in- cubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with pre- viously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.

  14. A method for rapid isolation of total RNA of high purity and yield from Arthrospira platensis.

    PubMed

    Pathak, Ravi Ramesh; Lochab, Sunila

    2010-07-01

    Arthrospira (Spirulina) platensis is widely used as a food supplement and has been an economically important species for centuries. However, the genetic aspect of studies of this particular organism has always been neglected, mainly because of the nonavailability of suitable methods for isolation of nucleic acids and the difficulties faced during further manipulations. Although total RNA has been isolated using commercially available kits, we present a method optimized to obtain DNA-free total RNA of higher yields and higher purity in less time than is required by other methods (<2 h). It involves hot phenol - chloroform - IAA extraction using an aqueous to organic phase ratio of 1:2 followed by lithium chloride precipitation and 70% ethanol wash. This method, optimized for the cyanobacterium Arthrospira (Spirulina) platensis, eliminates the need for DNase treatment and produces high-quality RNA, as validated by bioanalyzer, RT-PCR, and cloning. With the recent release of the Arthrospira genome, the current method will be of great value for carrying out high-throughput studies like microarray and real-time PCR. PMID:20651857

  15. Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material

    PubMed Central

    El-Khoury, Victoria; Pierson, Sandrine; Kaoma, Tony; Bernardin, François; Berchem, Guy

    2016-01-01

    MicroRNAs (miRNAs) have emerged as promising cancer biomarkers. However, exploiting their informative potential requires careful optimization of their detection. Here, we compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling. We used increasing amounts of starting material to investigate the impact of the input material size on miRNA extraction. We showed that miRNA recovery was largely influenced by the isolation method and by the amount of input material. In particular, the miRCURY™ kit provided highly pure RNA. However, its columns poorly recovered miRNAs from limiting amounts of cells and plasma, and rapidly saturated by large RNA species and plasma components, thus impeding miRNA recovery from high input amounts. Overall, the miRNeasy® kit permitted a better miRNA detection despite a less pure extracted RNA. Nevertheless, some miRNAs were preferentially or exclusively isolated by either of the methods. Trizol® LS resulted in very low purity RNA which affected RT-qPCR efficiency. In general, miRCURY™ biofluids kit efficiently extracted miRNAs from plasma. A careful selection of the RNA isolation method and the consideration of the type and size of input material are highly recommended to avoid biased results. PMID:26787294

  16. Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method.

    PubMed

    Enderle, Daniel; Spiel, Alexandra; Coticchia, Christine M; Berghoff, Emily; Mueller, Romy; Schlumpberger, Martin; Sprenger-Haussels, Markus; Shaffer, Jonathan M; Lader, Eric; Skog, Johan; Noerholm, Mikkel

    2015-01-01

    Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as "exoRNeasy Serum/Plasma Maxi Kit". PMID:26317354

  17. Isolation of total RNA from pollens.

    PubMed

    Bijli, K M; Singh, B P; Sridhara, S; Arora, N

    2001-05-01

    Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens. PMID:11426703

  18. Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

    PubMed Central

    Seneshaw, Mulugeta; Mirshahi, Faridoddin; Min, Hae-Ki; Asgharpour, Amon; Mirshahi, Shervin; Daita, Kalyani; Boyett, Sherry; Santhekadur, Prasanna K.; Fuchs, Michael; Sanyal, Arun J.

    2016-01-01

    Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases. PMID:27501005

  19. A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol.

    PubMed

    Likhite, Neah; Warawdekar, Ujjwala M

    2011-04-01

    The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at -80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction. PMID:21455480

  20. A Unique Method for Isolation and Solubilization of Proteins after Extraction of RNA from Tumor Tissue Using Trizol

    PubMed Central

    Likhite, Neah; Warawdekar, Ujjwala M.

    2011-01-01

    The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at −80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction. PMID:21455480

  1. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.

    PubMed

    Chomczynski, Piotr; Sacchi, Nicoletta

    2006-01-01

    Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years. PMID:17406285

  2. A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria.

    PubMed

    Sung, Kidon; Khan, Saeed A; Nawaz, Mohamed S; Khan, Ashraf A

    2003-12-01

    A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments. PMID:14659548

  3. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation maximum yield of highly-pure RNA from plant tissues rich in complex polysaccharides, polyphen...

  4. LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues

    PubMed Central

    2012-01-01

    Background Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium. Findings We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids. Conclusions The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures. PMID:22260178

  5. RNA isolation and fractionation with compaction agents

    NASA Technical Reports Server (NTRS)

    Murphy, J. C.; Fox, G. E.; Willson, R. C.

    2001-01-01

    A new approach to the isolation of RNA from bacterial lysates employs selective precipitation by compaction agents, such as hexammine cobalt and spermidine. Using 3.5 mM hexammine cobalt, total RNA can be selectively precipitated from a cell lysate. At a concentration of 2 mM hexammine cobalt, rRNA can be fractionated from low molecular weight RNA. The resulting RNA mixture is readily resolved to pure 5S and mixed 16S/23S rRNA by nondenaturing anion-exchange chromatography. Using a second stage of precipitation at 8 mM hexammine cobalt, the low molecular weight RNA fraction can be isolated by precipitation. Compaction precipitation was also applied to the purification of an artificial stable RNA derived from Escherichia coli 5S rRNA and to the isolation of an Escherichia coli-expressed ribozyme. Copyright 2001 Academic Press.

  6. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae.

    PubMed

    He, Yanxia; Guo, Xianguang; Xiang, Shifei; Li, Jiao; Li, Xiaoqin; Xiang, Hui; He, Jinlei; Chen, Dali; Chen, Jianping

    2016-07-01

    The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae. PMID:27147066

  7. Isolation of human immunodeficiency virus type 1 (HIV-1) RNA from feces by a simple method and difference between HIV-1 subpopulations in feces and serum.

    PubMed Central

    van der Hoek, L; Boom, R; Goudsmit, J; Snijders, F; Sol, C J

    1995-01-01

    A simple method for the isolation and subsequent detection of human immunodeficiency virus type 1 (HIV-1) RNA from feces is described. Viral RNA was isolated by the method developed by Boom et al. (R. Boom, C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990), which was adapted for feces. HIV-1 RNA was detected by reverse transcription (RT) followed by a nested PCR encompassing the V3 region. Reconstruction experiments revealed that the efficiencies of the extraction technique and the subsequent RT-PCR were not considerably affected by the varied composition of feces. The method was applied on fecal specimens from 18 HIV-1-infected individuals, among which were samples that had been stored for 9 years. It appeared that HIV-1 RNA was detectable in the feces of 12 persons (67%). Viral RNA was present in the feces of persons who fulfilled the criteria for CDC class II and CDC class III HIV infection as well as in patients who were diagnosed with AIDS (CDC class IV). Direct sequencing of amplimers obtained from paired fecal and serum specimens showed that differences in sequence heterogeneity existed. In one patient a remarkable difference in the HIV-1 sequences between isolates from feces and serum was observed. In conclusion, HIV-1 RNA is frequently present in the feces of HIV-1-infected individuals, and in some cases the HIV-1 subpopulation in feces differs from the HIV-1 subpopulation in serum. PMID:7751361

  8. Protocol for miRNA isolation from biofluids.

    PubMed

    Lekchnov, Evgeny A; Zaporozhchenko, Ivan A; Morozkin, Evgeny S; Bryzgunova, Olga E; Vlassov, Valentin V; Laktionov, Pavel P

    2016-04-15

    MicroRNAs (miRNAs) have been identified as promising biomarkers in cancer and other diseases. Packaging of miRNAs into vesicles and complexes with proteins ensures their stability in biological fluids but also complicates their isolation. Conventional protocols used to isolate cell-free RNA are generally successful in overcoming these difficulties; however, they are costly, labor-intensive, or heavily reliant on the use of hazardous chemicals. Here we describe a protocol that is suitable for isolating miRNAs from biofluids, including blood plasma and urine. The protocol is based on precipitation of proteins, denaturation of miRNA-containing complexes with octanoic acid and guanidine isothiocyanate, and subsequent purification of miRNA on spin columns. The efficacy of miRNA extraction by phenol-chloroform extraction, miRCURY RNA isolation kit--biofluids (Exiqon), and the proposed protocol was compared by quantitative reverse-transcription PCR of miR-16 and miR-126. The proposed protocol was slightly more effective for isolating miRNA from plasma and significantly superior to the other two methods for miRNA isolation from urine. Spectrophotometry and SDS-PAGE data suggest that the disparity in performance between miRCURY Biofluids and the proposed protocol can be attributed to differences in precipitation mechanisms, as confirmed by the retention of different proteins in the supernatant. PMID:26874020

  9. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    PubMed Central

    Fagotti, A; Gabbiani, G; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa. PMID:9518500

  10. Isolation of full-length RNA from a thermophilic cyanobacterium.

    PubMed

    Luo, X Z; Stevens, S E

    1997-11-01

    Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate. PMID:9383558

  11. Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil.

    PubMed

    Dineen, Shauna M; Aranda, Roman; Dietz, Marianne E; Anders, Douglas L; Robertson, James M

    2010-09-01

    Nucleic acid extraction is a critical step in the detection of an unknown biological agent. However, success can vary depending on the isolation and identification methods chosen and the difficulty of extraction from environmental matrices. In this work, bacteriophage MS2 RNA was extracted from three soil matrices, sand, clay, and loam, using five commercially available kits: the PowerSoil Total RNA Isolation, E.Z.N.A. Soil RNA, FastRNA Pro Soil-Direct, FastRNA Pro Soil-Indirect, and IT 1-2-3 Platinum Path kits. Success of the isolation was determined using an MS2-specific RT-PCR assay. The reproducibility and sensitivity of each method in the hands of both experienced and novice users were assessed and compared. Cost, operator time, and storage conditions were also considered in the evaluation. The RNA isolation method that yielded the best results, as defined by reproducibility and sensitivity, was the E.Z.N.A. Soil RNA kit for sand, the IT 1-2-3 Platinum Path Sample Purification kit for clay, and the FastRNA Pro Soil-Indirect kit for loam. However, if time and storage conditions are important considerations, the IT 1-2-3 Platinum Path kit may be appropriate for use with all soils since the kit has the shortest processing time and fewest temperature requirements. PMID:20417664

  12. mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

    PubMed Central

    Jerome, J F; Jaehning, J A

    1986-01-01

    We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II. Images PMID:3537708

  13. Isolation of three testis-specific genes (TSA303, TSA806, TSA903) by a differential mRNA display method

    SciTech Connect

    Ozaki, Kouichi; Kuroki, Tamotsu; Hayashi, Seitaku; Nakamura, Yusuke

    1996-09-01

    We isolated three human testis-specific genes by a differential mRNA display method. The cDNAs contained open reading frames of 1620, 453, and 333 nucleotides, encoding 540, 151, 111 amino acids, respectively. The first of these genes, designated TSA303, encodes a novel protein homologous to TCP20, one of the subunits of the human TRiC chaperonin complex that can bind newly synthesized or unstable folding intermediates of polypeptides and assist substrate proteins in folding, assembly, and transport. The second, TSA806, encodes a novel protein containing 3.3 contiguous repeats of the cdc10/swi6 (ankyrin) motif that was originally found in products of cell cycle control genes of yeast and cell fate determination genes in Drosophila and Caenorhabditis elegans. The third gene, TSA903, encodes a protein homologous to the C-terminal region of murine uridine monophosphate kinase. Northern blot analysis confirmed that in 16 human adult tissues examined, each of these genes was expressed specifically in the testis. From the results of cDNA screening of nearly 1 million plaques, the abundance of each transcript in a preparation of total mRNA was estimated as 0.0004% (TSA303), 0.0006% (TSA806), and 0.0002% (TSA903). Our results imply that the differential display method is a powerful tool for isolation of tissue-specific genes even if they are expressed at a level as low as 1 in several hundred thousand to a million molecules of total mRNA. 38 refs., 1 fig., 3 tabs.

  14. New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

    PubMed Central

    Kawashima, Kenji; Lucero, Ginger; Ackermann, Mark R.

    2005-01-01

    We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead. PMID:16136226

  15. Isolation of mRNA from specific tissues of Drosophila by mRNA tagging.

    PubMed

    Yang, Zhiyong; Edenberg, Howard J; Davis, Ronald L

    2005-01-01

    To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes. PMID:16204451

  16. Polysome Preparation, RNA Isolation and Analysis

    PubMed Central

    Zhang, Hailong; Zhou, Muxiang

    2016-01-01

    During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosome can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.

  17. Method for isolating nucleic acids

    DOEpatents

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  18. Usefulness of the ID32 staph system and a method based on rRNA gene restriction site polymorphism analysis for species and subspecies identification of staphylococcal clinical isolates.

    PubMed Central

    Chesneau, O; Aubert, S; Morvan, A; Guesdon, J L; el Solh, N

    1992-01-01

    The usefulness of the ID32 Staph System and a method based on rRNA gene restriction site polymorphism was evaluated by the study of 42 staphylococcal clinical isolates phenotypically difficult to identify. The ID32 Staph micromethod and the genomic method are adapted for recognition of 27 and 31 staphylococcal taxa, respectively. The genomic method is based on a Dice analysis of the hybridization patterns obtained by cutting the cellular DNA either with EcoRI or with HindIII and by probing with pBA2, containing the Bacillus subtilis gene encoding 16S rRNA, labeled either with [alpha-32P]dCTP or with acetylaminofluorene. This study showed that the nonradioactive labeling provided a better resolution of the hybridizing bands than radioactive labeling. Of the 42 isolates selected, only 22 could be assigned to a staphylococcal species by the ID32 Staph System, whereas 35 could be identified by the genomic method. This latter method also enabled the screening of three unclassified isolates having hybridization patterns more closely related to each other than to any of the 31 staphylococcal taxa investigated. These three isolates could belong to a staphylococcal taxon not yet described. Images PMID:1357001

  19. Identification of Clinical Coryneform Bacterial Isolates: Comparison of Biochemical Methods and Sequence Analysis of 16S rRNA and rpoB Genes▿

    PubMed Central

    Adderson, Elisabeth E.; Boudreaux, Jan W.; Cummings, Jessica R.; Pounds, Stanley; Wilson, Deborah A.; Procop, Gary W.; Hayden, Randall T.

    2008-01-01

    We compared the relative levels of effectiveness of three commercial identification kits and three nucleic acid amplification tests for the identification of coryneform bacteria by testing 50 diverse isolates, including 12 well-characterized control strains and 38 organisms obtained from pediatric oncology patients at our institution. Between 33.3 and 75.0% of control strains were correctly identified to the species level by phenotypic systems or nucleic acid amplification assays. The most sensitive tests were the API Coryne system and amplification and sequencing of the 16S rRNA gene using primers optimized for coryneform bacteria, which correctly identified 9 of 12 control isolates to the species level, and all strains with a high-confidence call were correctly identified. Organisms not correctly identified were species not included in the test kit databases or not producing a pattern of reactions included in kit databases or which could not be differentiated among several genospecies based on reaction patterns. Nucleic acid amplification assays had limited abilities to identify some bacteria to the species level, and comparison of sequence homologies was complicated by the inclusion of allele sequences obtained from uncultivated and uncharacterized strains in databases. The utility of rpoB genotyping was limited by the small number of representative gene sequences that are currently available for comparison. The correlation between identifications produced by different classification systems was poor, particularly for clinical isolates. PMID:18160450

  20. Isolation of high-quality RNA from Reaumuria soongorica, a desert plant rich in secondary metabolites.

    PubMed

    Wang, Xiaohua; Xiao, Honglang; Chen, Guoxiong; Zhao, Xin; Huang, Chenghong; Chen, Cuiyun; Wang, Fang

    2011-06-01

    RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable. PMID:21136208

  1. RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

    PubMed Central

    Azevedo-Pouly, Ana Clara P.; Elgamal, Ola A.; Schmittgen, Thomas D.

    2015-01-01

    Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable. PMID:25145327

  2. Chromatin Isolation by RNA Purification (ChIRP)

    PubMed Central

    Chu, Ci; Quinn, Jeffrey; Chang, Howard Y.

    2012-01-01

    Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13. Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification

  3. Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

    PubMed

    Bell, Thomas J; Eberwine, James

    2015-01-01

    RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data. PMID:26202289

  4. Rapid isolation of mycoviral double-stranded RNA from Botrytis cinerea and Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA. Results A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing. Conclusions The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA. PMID:21262001

  5. Isolation of High-Quality Total RNA from Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook).

    PubMed

    Ma, Zhihui; Huang, Binlong; Xu, Shanshan; Chen, Yu; Li, Shubin; Lin, Sizu

    2015-01-01

    RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method. PMID:26083257

  6. Isolation of quality total RNA from the aromatic plant Origanum onites.

    PubMed

    Poyraz, Ismail; Sözen, Emel; Arslanyolub, Muhittin

    2010-01-01

    We successfully used the guanidine isothiocyanate method for isolation of total RNA from leaf, stem, and root tissues of the aromatic plant Origanum onites. The RNA was extracted with TRI Reagent at room temperature and was recovered by isopropanol precipitation. The isolated RNA was capable of reverse transcription. The extraction method described here does not require ultracentrifugation, and it is fast, simple, and effective. The procedure can be completed within 3 hours and may be applicable to other aromatic medicinal plants containing high amounts of phenolic compounds. PMID:20469647

  7. Evaluations of methods for the isolation of high quality RNA from bovine and cervine hide biopsies for use in gene expression studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data pr...

  8. Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile.

    PubMed

    Li, Ling; Piontek, Klaus B; Kumbhari, Vivek; Ishida, Masaharu; Selaru, Florin M

    2016-01-01

    Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses. Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 °C. PMID:27341293

  9. Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium

    SciTech Connect

    Brown, J.W.; Reeve, J.N.

    1986-05-01

    Polyadenylated (poly(A)/sup +/) RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4/sup 0/C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)/sup +/ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)/sup +/ RNAs are unstable.

  10. [Characteristics of virus double-stranded RNA, isolated from microscopic fungi parasitizing on sugar beet].

    PubMed

    Mel'nychuk, M D; Spyrydonov, V H; Oleksiienko, I P

    2005-01-01

    We have carried out comparative studies of double-stranded RNA (dsRNA) of viral nature isolated from sugar beet leaves and from mycelium of microscopic fungi using different methods such as PAAG electrophoresis and by polymerase chain reaction (PCR). It was shown that the fragments of dsRNA from sugar beet leaves and from mycelium microscopic fungi had the identical electrophoretic pattern and the same size (1.8 and 2.0 kbp). Using PCR technique it was shown, that isolated dsRNA have a common template for amplification. Electron microscopy of PCR-positive mycelium allows us to detect the virus particles of the spherical form with diameter 30-40 nm. The obtained data confirm our previous suppositions, concerning the belonging of isolated dsRNAs (size 1.8 and 2.0 kbp) to new mycovirus targeted a microscopic fungus, instead of beet cryptic viruses. PMID:16250236

  11. Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

    PubMed Central

    Yu, Claire; Young, Stuart; Russo, Valerio; Amsden, Brian G.

    2013-01-01

    Extracting high-quality RNA from hydrogels containing polysaccharide components is challenging, as traditional RNA isolation techniques designed for cells and tissues can have limited yields and purity due to physiochemical interactions between the nucleic acids and the biomaterials. In this study, a comparative analysis of several different RNA isolation methods was performed on human adipose-derived stem cells photo-encapsulated within methacrylated glycol chitosan hydrogels. The results demonstrated that RNA isolation methods with cetyl trimethylammonium bromide (CTAB) buffer followed by purification with an RNeasy® mini kit resulted in low yields of RNA, except when the samples were preminced directly within the buffer. In addition, genomic DNA contamination during reverse transcriptase–polymerase chain reaction (RT-PCR) analysis was observed in the hydrogels processed with the CTAB-based methods. Isolation methods using TRIzol® in combination with one of a Qiaex® gel extraction kit, an RNeasy® mini kit, or an extended solvent purification method extracted RNA suitable for gene amplification, with no evidence of genomic contamination. The latter two methods yielded the best results in terms of yield and amplification efficiency. Predigestion of the scaffolds with lysozyme was investigated as a possible means of enhancing RNA extraction from the polysaccharide gels, with no improvements observed in terms of the purity, yield, or amplification efficiency. Overall, this work highlights the application of a TRIzol®+extended solvent purification method for optimizing RNA extraction that can be applied to obtain reliable and accurate gene expression data in studies investigating cells seeded in chitosan-based scaffolds. PMID:23448167

  12. Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation.

    PubMed

    Bai, Baoyan; Laiho, Marikki

    2016-01-01

    The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments. PMID:27576723

  13. Isolation of functional RNA from plant tissues rich in phenolic compounds.

    PubMed

    Schneiderbauer, A; Sandermann, H; Ernst, D

    1991-08-15

    A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation. PMID:1719845

  14. RNA synthesis in isolated rat osteoclasts: inhibitory effect of calcitonin.

    PubMed

    Zheng, M H; Papadimitriou, J M; Nicholson, G C

    1991-01-01

    The metabolism of RNA has not been studied in the osteoclast (OC) because these bone-resorbing cells are only available in small numbers and cultures are always contaminated with other cells. Using two single-cell assay techniques, tritiated uridine (3H-UdR) autoradiography and gallocyanin quantitative cytophotometry, we have examined RNA synthesis in OCs isolated from neonatal rats. Oligo-nuclear OCs showed greater nuclear uptake of 3H-UdR than cells with many nuclei, and the variance of nuclear labeling within polykarya was greater in the latter, possibly because they contain nuclei of various ages. Salmon calcitonin (sCT) was a potent (ED50 approximately 5 x 10(-12) M) and rapid (40% reduction in 2 h, 75% reduction in 6 h) inhibitor of 3H-UdR uptake, and also reduced cytochemical total cellular RNA by 22% within 4 h. Forskolin (10(-5) M) inhibited nuclear uptake of 3H-UdR, suggesting that the sCT response may be mediated by cyclic AMP. Following a short (30 min) exposure to sCT, there was a progressive decline in labeling, followed by complete recovery by 4.5 h, a response possibly related to the phenomenon of calcitonin-induced persistent activation of adenylate cyclase. Inhibition of OC RNA synthesis may be an important component of its anti-resorptive action. PMID:1723609

  15. Torsional vibration isolator and method

    SciTech Connect

    Allen, C.A.; Durrett, V.D.

    1986-10-21

    This patent describes a multicylinder internal combustion engine having a rotatable crankshaft and a rotatable flywheel which together define an inertial system rotating about a predetermined axis of rotation. An improvement is described here which facilitates avoiding destructive effects on the crankshaft of stress induced by torsional vibration. The method comprises an elastomeric annulus coupling means operatively interposed between the crankshaft and flywheel for coupling the crankshaft and flywheel together for rotation of the flywheel with the crankshaft. The coupling means has a torsional spring rate of less than 20,000 in lb/radian effective to permit substantial angular displacement between the flywheel and the crankshaft for isolating the rotating inertia of the flywheel from the rotating inertia of the crankshaft after engine startup. The coupling means avoids dampening while preventing torsional vibration from being transferred between the flywheel and the crankshaft.

  16. Multiscale methods for computational RNA enzymology

    PubMed Central

    Panteva, Maria T.; Dissanayake, Thakshila; Chen, Haoyuan; Radak, Brian K.; Kuechler, Erich R.; Giambaşu, George M.; Lee, Tai-Sung; York, Darrin M.

    2016-01-01

    RNA catalysis is of fundamental importance to biology and yet remains ill-understood due to its complex nature. The multi-dimensional “problem space” of RNA catalysis includes both local and global conformational rearrangements, changes in the ion atmosphere around nucleic acids and metal ion binding, dependence on potentially correlated protonation states of key residues and bond breaking/forming in the chemical steps of the reaction. The goal of this article is to summarize and apply multiscale modeling methods in an effort to target the different parts of the RNA catalysis problem space while also addressing the limitations and pitfalls of these methods. Classical molecular dynamics (MD) simulations, reference interaction site model (RISM) calculations, constant pH molecular dynamics (CpHMD) simulations, Hamiltonian replica exchange molecular dynamics (HREMD) and quantum mechanical/molecular mechanical (QM/MM) simulations will be discussed in the context of the study of RNA backbone cleavage transesterification. This reaction is catalyzed by both RNA and protein enzymes, and here we examine the different mechanistic strategies taken by the hepatitis delta virus ribozyme (HDVr) and RNase A. PMID:25726472

  17. TECHNICAL BRIEF: Optimized pipeline for isolation of high-quality RNA from corneal cell subpopulations

    PubMed Central

    Bath, Chris; Fink, Trine; Vorum, Henrik; Hjortdal, Jesper

    2014-01-01

    Purpose: Attempts to determine the transcriptional profile of discrete subsets of limbal epithelial cells in situ using laser capture microdissection (LCM) face two major challenges. First, the transcriptional profile of cells within a tissue may rapidly change as the tissue is excised and exposed to cold ischemia. Second, there is a risk of degradation of the RNA as the cellular compartment is separated from the remaining tissue. An optimized protocol for LCM of corneal epithelium is presented to address these issues. Methods: Experiments using porcine eye globes were carried out to determine both optimal procedures and settings for tissue harvest, transport, storage, histology, LCM, and RNA isolation. The optimized protocol was validated using human corneal epithelium. Results: To facilitate preservation of the gene expression profile, we have developed a mechanical tool for dissection of cornea that, in combination with flash freezing, enables tissue to be stored within 5 min of enucleation of the eye. Furthermore, we describe how RNA from limbal crypt cells may be obtained using a procedure involving cryosectioning, histological staining, and LCM. Conclusion: In this paper, we describe an optimized method for isolating high-quality RNA from cellular subpopulations confined to the limbal crypts of the cornea. The procedure yields RNA in amounts and quality suitable for downstream gene expression analyses, such as microarrays or next generation sequencing. PMID:24940035

  18. Isolation of high quality RNA from pistachio (Pistacia vera L.) and other woody plants high in secondary metabolites.

    PubMed

    Moazzam Jazi, Maryam; Rajaei, Saideh; Seyedi, Seyed Mahdi

    2015-10-01

    The quality and quantity of RNA are critical for successful downstream transcriptome-based studies such as microarrays and RNA sequencing (RNA-Seq). RNA isolation from woody plants, such as Pistacia vera, with very high amounts of polyphenols and polysaccharides is an enormous challenge. Here, we describe a highly efficient protocol that overcomes the limitations posed by poor quality and low yield of isolated RNA from pistachio and various recalcitrant woody plants. The key factors that resulted in a yield of 150 μg of high quality RNA per 200 mg of plant tissue include the elimination of phenol from the extraction buffer, raising the concentration of β-mercaptoethanol, long time incubation at 65 °C, and nucleic acid precipitation with optimized volume of NaCl and isopropyl alcohol. Also, the A260/A280 and A260/A230 of extracted RNA were about 1.9-2.1and 2.2-2.3, respectively, revealing the high purity. Since the isolated RNA passed highly stringent quality control standards for sensitive reactions, including RNA sequencing and real-time PCR, it can be considered as a reliable and cost-effective method for RNA extraction from woody plants. PMID:26600686

  19. Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

    PubMed Central

    Schiebel, W; Pélissier, T; Riedel, L; Thalmeir, S; Schiebel, R; Kempe, D; Lottspeich, F; Sänger, H L; Wassenegger, M

    1998-01-01

    A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. PMID:9836747

  20. Isolation and analysis of mRNA from environmental microbial communities.

    PubMed

    McGrath, Ken C; Thomas-Hall, Skye R; Cheng, Chu Ting; Leo, Lesina; Alexa, Andrei; Schmidt, Susanne; Schenk, Peer M

    2008-10-01

    The advent of metagenomics has revealed that our planet harbors millions of previously undiscovered microbial species. However, functional insights into the activities of microbial communities cannot easily be obtained using metagenomics. Using transcriptional analyses to study microbial gene functions is currently problematic due to difficulties working with unstable microbial mRNA as a small fraction of total cellular RNA. Current techniques can be expensive and time consuming, and still result in significant levels of rRNA contamination. We have adapted techniques to rapidly isolate high high-quality RNA from environmental samples and developed a simple method for specific isolation of mRNA by size separation. This new technique was evaluated by constructing cDNA libraries directly from uncultured environmental microbial communities, including agricultural soil samples, aquatic flocculants, organic composts, mammalian oral and faecal samples, and wastewater sludge. The sequencing of a fraction of these cDNA clones revealed a high degree of novelty, demonstrating the potential of this approach to capture a large number of unique transcripts directly from the environment. To our knowledge, this is the first study that uses gel electrophoresis to isolate mRNA from microbial communities. We conclude that this method could be used to provide insights into the microbial 'metatranscriptome' of entire microbial communities. Coupled with high-throughput sequencing or the construction of cDNA microarrays, this approach will provide a useful tool to study the transcriptional activities of microorganisms, including those of entire microbial communities and of non-culturable microorganisms. PMID:18582973

  1. Aphid Transmission Alters the Genomic and Defective RNA Populations of Citrus tristeza virus Isolates.

    PubMed

    Albiach-Martí, M R; Guerri, J; de Mendoza, A H; Laigret, F; Ballester-Olmos, J F; Moreno, P

    2000-02-01

    ABSTRACT A total of 14 Spanish isolates of Citrus tristeza virus (CTV) and 1 isolate from Japan were transmitted by Aphis gossypii, and the subisolates obtained were compared with the source isolates for symptom expression and double-stranded RNA (dsRNA) pattern. Of the 14 Spanish isolates, 9 showed altered dsRNA patterns after aphid transmission but only minor variations in the intensity of symptoms induced on Mexican lime. Northern blot hybridization with complementary DNA (cDNA) probes corresponding to both the 5' and the 3' termini of the CTV genomic RNA (gRNA) showed that the dsRNA bands that could be used to discriminate between the dsRNA pattern of the source and the aphid-transmitted isolates were the replicative forms of defective RNAs (D-RNAs). Conversely, the Japanese isolate and two subisolates obtained from it by aphid transmission had the same dsRNA pattern, but one of the subisolates induced milder symptoms in several hosts. Dot-blot hybridization with cDNA probes representing several regions of the gRNA showed that most of the aphid-transmitted isolates differed from the corresponding source isolate by their hybridization pattern. Our results indicate that aphid transmission often sorts the populations of gRNA variants and D-RNAs present in CTV isolates. PMID:18944601

  2. An improved method for extraction of high-quality total RNA from oil seeds.

    PubMed

    Rayani, Azadeh; Dehghan Nayeri, Fatemeh

    2015-04-01

    Seeds of oilseed plants that contain large amounts of oil, polysaccharides, proteins and polyphenols are not amenable to conventional RNA isolation protocols. The presence of these substances affects the quality and quantity of isolated nucleic acids. Here, a rapid and efficient RNA isolation protocol that, in contrast to other methods tested, allows high purify, integrity and yield of total RNA from seeds of sesame, corn, sunflower, flax and rapeseed was developed. The average yields of total RNA from 70 mg oil seeds ranged from 84 to 310 µg with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative RT-PCR, cDNA synthesis and expression analysis. PMID:25534638

  3. RNA-Seq Library Generation from Rare Human Cells Isolated by FACS

    PubMed Central

    Gkountela, Sofia; Clark, Amander T.

    2016-01-01

    High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16–16.5 weeks of development and 4,000 sorted hESCs (n = 3). We predict that multiplexed libraries can also be generated by replacing the single-plex 3′ adapter used here with a multiplexing compatible 3′ adapter and indexed PCR primers.

  4. Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine.

    PubMed

    Crossland, Rachel E; Norden, Jean; Bibby, Louis A; Davis, Joanna; Dickinson, Anne M

    2016-02-01

    MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the

  5. RNASwift: A rapid, versatile RNA extraction method free from phenol and chloroform.

    PubMed

    Nwokeoji, Alison O; Kilby, Peter M; Portwood, David E; Dickman, Mark J

    2016-11-01

    RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 μg of RNA from only 10(8)Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods. PMID:27495141

  6. Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

    PubMed

    Lange, Carla C; Brito, Maria A V P; Reis, Daniele R L; Machado, Marco A; Guimarães, Alessandro S; Azevedo, Ana L S; Salles, Érica B; Alvim, Mariana C T; Silva, Fabiana S; Meurer, Igor R

    2015-04-17

    Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms. PMID:25704228

  7. Characterization of the partial RNA1 and RNA2 3' untranslated region of tomato ringspot virus isolates from North America

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 3' non-translated regions (NTRs) of RNA1 and RNA2 of Tomato ringspot virus (ToRSV) are long and virtually identical. In this study, sequences containing most of the 3’ NTRs (1168-1265 bp) were determined from 18 ToRSV isolates collected from fruit trees, small fruits, and grapevines in North Am...

  8. The paraffin-embedded RNA metric (PERM) for RNA isolated from formalin-fixed, paraffin-embedded tissue.

    PubMed

    Chung, Joon-Yong; Cho, Hanbyoul; Hewitt, Stephen M

    2016-01-01

    RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue is commonly evaluated in both investigative and diagnostic pathology. However, the quality of the data is directly impacted by RNA quality. The RNA integrity number (RIN), an algorithm based on a combination of electrophoretic features, is widely applied to RNA isolated from paraffin-embedded tissue, but it is a poor indicator of the quality of that RNA. Here we describe the novel paraffin-embedded RNA metric (PERM) for quantifying the quality of RNA from FFPE tissue. The PERM is based on a formula that approximates a weighted area-under-the-curve analysis of an electropherogram of the extracted RNA. Using biochemically degraded RNAs prepared from experimentally fixed mouse kidney specimens, we demonstrate that PERM values correlate with mRNA transcript measurements determined using the QuantiGene system. Furthermore, PERM values correlate with real-time PCR data. Our results demonstrate that the PERM can be used to qualify RNA for different end-point studies and may be a valuable tool for molecular studies using RNA extracted from FFPE tissue. PMID:27177816

  9. Isolation of RNA from field-grown jute (Corchorus capsularis) plant in different developmental stages for effective downstream molecular analysis.

    PubMed

    Samanta, Pradipta; Sadhukhan, Sanjoy; Das, Subrata; Joshi, Alpana; Sen, Soumitra K; Basu, Asitava

    2011-10-01

    Jute (Corchorus capsularis), as a natural fibre producing plant species, ranks next to cotton only. Today, biotechnological approach has been considered as most accepted means for any genetic improvement of plant species. However, genetic control of the fibre development in jute has not yet been explored sufficiently for desired genetic improvement. One of the major impediments in exploring the genetic architecture in this crop at molecular level is the availability of good quality RNA from field-grown plant tissues mostly due to the presence of high amount of mucilage and phenolics. Development of a suitable RNA isolation method is becoming essential for deciphering developmental stage-specific gene expression pattern related to fibre formation in this crop species. A combination of modified hot borate buffer followed by isopycnic centrifugation (termed as HBIC) was adopted and found to be the best isolation method yielding sufficient quantity (~350-500 μg/gm fresh tissue) and good quality (A(260/280) ratio 1.88 to 1.91) RNA depending on the developmental stage of stem tissue from field-grown jute plant. The poly A(+) RNA purified from total RNA isolated by the present method was found amenable to efficient RT-PCR and cDNA library construction. The present development of RNA isolation was found to be appropriate for gene expression analysis related to fibre formation in this economically important jute plant in near future. PMID:21327574

  10. Assessing Social Isolation: Pilot Testing Different Methods.

    PubMed

    Taylor, Harry Owen; Herbers, Stephanie; Talisman, Samuel; Morrow-Howell, Nancy

    2016-04-01

    Social isolation is a significant public health problem among many older adults; however, most of the empirical knowledge about isolation derives from community-based samples. There has been less attention given to isolation in senior housing communities. The objectives of this pilot study were to test two methods to identify socially isolated residents in low-income senior housing and compare findings about the extent of isolation from these two methods. The first method, self-report by residents, included 47 out of 135 residents who completed in-person interviews. To determine self-report isolation, residents completed the Lubben Social Network Scale 6 (LSNS-6). The second method involved a staff member who reported the extent of isolation on all 135 residents via an online survey. Results indicated that 26% of residents who were interviewed were deemed socially isolated by the LSNS-6. Staff members rated 12% of residents as having some or a lot of isolation. In comparing the two methods, staff members rated 2% of interviewed residents as having a lot of isolation. The combination of self-report and staff report could be more informative than just self-report alone, particularly when participation rates are low. However, researchers should be aware of the potential discrepancy between these two methods. PMID:27276687

  11. Methods for Characterization of Alternative RNA Splicing

    PubMed Central

    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. PMID:26721495

  12. Anti-tumour cytotoxicity of poly(A)-containing messenger RNA isolated from tumour-specific immunogenic RNA.

    PubMed

    Greenup, C J; Vallera, D A; Pennline, K J; Kolodziej, B J; Dodd, M C

    1978-07-01

    The transfer of tumour-specific cytotoxicity against a murine fibrosar-coma has been demonstrated in vitro using xenogeneic RNA extracted from tumour-cell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA. Treated cells were tested in vitro using an adaptation of the Takasugi and Klein microcytotoxicity assay. Percent cytotoxicity was calculted using cells treated with fractions of normal RNA as control. An increase in tumour cytotoxicity was found with poly(A)-containing immune RNA. The optimum dose of poly(A)-tailed immune RNA was estimated as 6.5 microgram of RNA per 4 x 10(6) lymphocytes. Populations of lymphocytes were separated using glass and nylon wool. T- and B-enriched populations were treated with various RNA components. The adherent cell population showed no significant cytotoxicity, whilst treatment of the nonadherent population with poly(A)-tailed immune RNA produced high levels of cytotoxicity. PMID:687518

  13. Anti-tumour cytotoxicity of poly(A)-containing messenger RNA isolated from tumour-specific immunogenic RNA.

    PubMed Central

    Greenup, C. J.; Vallera, D. A.; Pennline, K. J.; Kolodziej, B. J.; Dodd, M. C.

    1978-01-01

    The transfer of tumour-specific cytotoxicity against a murine fibrosar-coma has been demonstrated in vitro using xenogeneic RNA extracted from tumour-cell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA. Treated cells were tested in vitro using an adaptation of the Takasugi and Klein microcytotoxicity assay. Percent cytotoxicity was calculted using cells treated with fractions of normal RNA as control. An increase in tumour cytotoxicity was found with poly(A)-containing immune RNA. The optimum dose of poly(A)-tailed immune RNA was estimated as 6.5 microgram of RNA per 4 x 10(6) lymphocytes. Populations of lymphocytes were separated using glass and nylon wool. T- and B-enriched populations were treated with various RNA components. The adherent cell population showed no significant cytotoxicity, whilst treatment of the nonadherent population with poly(A)-tailed immune RNA produced high levels of cytotoxicity. PMID:687518

  14. Sweet potato feathery mottle potyvirus (C1 isolate) virion and RNA purification.

    PubMed

    Nakashima, J T; Salazar, L F; Wood, K R

    1993-09-01

    A procedure for the purification of a Peruvian isolate (C1) of sweet potato feathery mottle potyvirus (SPFMV) and infective RNA has been developed. The use of Hepes [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid] buffer containing urea and sodium EDTA as a base for tissue extraction and virus suspension enabled good yields of virus (35-50 mg/100 g) to be obtained from Nicotiana benthamiana L. Domin. A short RNA isolation procedure yielded infectious RNA, from which ds cDNA of nearly genome size could be obtained. Sweet potato feathery mottle potyvirus, Purification, RNA isolation, cDNA synthesis. PMID:8227274

  15. [Rapid method to extract high-quality RNA from activated sludge].

    PubMed

    Jin, Min; Zhao, Zu-Guo; Qiu, Zhi-Gang; Wang, Jing-Feng; Chen, Zhao-Li; Shen, Zhi-Qiang; Li, Chao; Wang, Xin-Wei; Dong, Yan; Li, Jun-Wen

    2010-01-01

    An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. That is, the precipitated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to direct lysis of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA, RNA purifying column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, can effectively extract high-quality RNA. It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA can be amplified by RT-PCR. Compared with other methods, it showed great advantage of low cost and high efficiency and can be applied to RNA extraction of activated sludge in a large number. Furthermore, T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods. This work established a rapid and effective method to extract high-quality RNA from activated sludge and would show great potential for monitoring microbial changes and studying metabolism and community array of activated sludge. PMID:20329549

  16. Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.

    PubMed

    Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius

    2015-05-01

    Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. PMID:25625965

  17. FLDS: A Comprehensive dsRNA Sequencing Method for Intracellular RNA Virus Surveillance.

    PubMed

    Urayama, Syun-Ichi; Takaki, Yoshihiro; Nunoura, Takuro

    2016-03-26

    Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein constructed an effective metagenomic method for RNA viruses by targeting long double-stranded (ds)RNA in cellular organisms, which is a hallmark of infection, or the replication of dsRNA and single-stranded (ss)RNA viruses, except for retroviruses. This novel dsRNA targeting metagenomic method is characterized by an extremely high recovery rate of viral RNA sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting RNA viruses. This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. Our approach will be a powerful tool for cataloging RNA viruses associated with organisms of interest. PMID:26877136

  18. FLDS: A Comprehensive dsRNA Sequencing Method for Intracellular RNA Virus Surveillance

    PubMed Central

    Urayama, Syun-ichi; Takaki, Yoshihiro; Nunoura, Takuro

    2016-01-01

    Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein constructed an effective metagenomic method for RNA viruses by targeting long double-stranded (ds)RNA in cellular organisms, which is a hallmark of infection, or the replication of dsRNA and single-stranded (ss)RNA viruses, except for retroviruses. This novel dsRNA targeting metagenomic method is characterized by an extremely high recovery rate of viral RNA sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting RNA viruses. This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. Our approach will be a powerful tool for cataloging RNA viruses associated with organisms of interest. PMID:26877136

  19. Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

    PubMed Central

    Flanegan, J B; Van Dyke, T A

    1979-01-01

    A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template. PMID:232168

  20. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam

    PubMed Central

    2013-01-01

    Background 16S rRNA methylase-producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed clinical strains of 16S rRNA methylase-producing Acinetobactor baumannii and Pseudomonas aeruginosa obtained from clinical isolates in medical settings in Vietnam. Methods From 2008 to 2011, 101 clinical strains of A. baumannii and 15 of P. aeruginosa were isolated from patients in intensive care units (ICUs) in two medical settings in Vietnam. Antimicrobial susceptibilities were determined using the microdilution method and epidemiological analysis was performed by pulsed-field gel electrophoresis and MLST. Genes encoding the 16S rRNA methylases, OXAs and CTX-Ms were analyzed by PCR and sequence analysis. Results 16S rRNA methylase-producing Gram-negative pathogens were detected in two hospitals in Vietnam. Of the 101 clinical isolates of A. baumannii and the 15 of P. aeruginosa isolated from two ICUs in these hospitals, 72 (71.3%) were highly resistant to amikacin, arbekacin and gentamicin, with MICs greater than 1,024 mg/L. The 16S rRNA methylases ArmA and RmtB were produced by 61 and 9 isolates of A. baumannii, respectively, and RmtB was produced by 2 isolates of P. aeruginosa. Moreover, 52 of the A. baumannii isolates producing 16S rRNA methylases harbored both blaOXA-23-like and blaOXA-51-like genes. Most A. baumannii isolates producing 16S rRNA methylase obtained in hospital A in Hanoi were ST91 and ST231, whereas most from hospital B in Ho Chi Minh City were ST136, ST195, and ST254. The two P. aeruginosa isolates harboring rmtB showed different patterns on PFGE, one each corresponding to ST217 and ST313. Conclusions Gram-negative bacteria producing the 16S rRNA methylases ArmA and RmtB are emerging in medical settings in Vietnam. A. baumannii isolates in northern and southern regions of Vietnam may be of different lineages. PMID:23721359

  1. Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia

    PubMed Central

    Al Sheikh, Yazeed A.; Marie, Mohammed Ali M.; John, James; Krishnappa, Lakshmana Gowda; Dabwab, Khaled Homoud M.

    2014-01-01

    Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were bla TEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae. PMID:25005152

  2. Evaluation of the semen swim-up method for bovine sperm RNA extraction.

    PubMed

    Han, C M; Chen, R; Li, T; Chen, X L; Zheng, Y F; Ma, M T; Gao, Q H

    2016-01-01

    Isolation of high-quality RNA is important for assessing sperm gene expression, and semen purification methods may affect the integrity of the isolated RNA. This study evaluated the effectiveness of the sperm swim-up method for seminal RNA isolation. Frozen semen samples in straws from three bulls of proven fertility were purified by the swim-up method. RNA extraction was carried out using the E.Z.N.A.(TM) Total RNA kit II, with non-swim-up sperm as a control. Total sperm RNA was analyzed by UV spectrophotometry, reverse transcription polymerase chain reaction (RT-PCR), and agarose gel electrophoresis, and expression of the sex-determining region on the Y chromosome (SRY), leptin (LEP), and ribosomal protein subunit 23 (RPS23) genes, were determined. 18S RNA was used as a positive control. Fewer somatic cells were found in sperm swim-up samples than in the non-swim-up counterparts (0 x 10(3) vs 17.33 ± 2.52 x 10(3) sperm, P < 0.05). In addition, high-quality RNA was obtained in about 2 h, with no significant difference between groups. Interestingly, the yields of RNA fragments containing ≥200 nucleotides were significantly reduced in sperm swim-up samples (0.92 ± 0.41 x 10(7) sperm) compared with the non-swim-up samples (1.36 ± 0.33 x 10(7) sperm, P < 0.05). After RT-PCR, clear bands representing SRY, LEP, and RPS23 in sperm cDNA were observed on agarose gel electrophoresis. Finally, no bands corresponding to 18S RNA were found in RNA samples from the sperm swim-up group. Our findings suggest that small amounts of sperm RNA can be efficiently extracted from frozen straw semen samples using the swim-up technique. PMID:27173315

  3. Current methods for the isolation of extracellular vesicles.

    PubMed

    Momen-Heravi, Fatemeh; Balaj, Leonora; Alian, Sara; Mantel, Pierre-Yves; Halleck, Allison E; Trachtenberg, Alexander J; Soria, Cesar E; Oquin, Shanice; Bonebreak, Christina M; Saracoglu, Elif; Skog, Johan; Kuo, Winston Patrick

    2013-10-01

    Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter. PMID:23770532

  4. A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA.

    PubMed

    Hillman, B I; Tian, Y; Bedker, P J; Brown, M P

    1992-03-01

    We have synthesized and mapped a cDNA library representing the one major dsRNA element associated with hypovirulence in strain NB58 of the chestnut blight fungus, Cryphonectira (=Endothia) parasitica, which was isolated from recovering chestnut trees in New Jersey, U.S.A. The linear dsRNA has a size of approximately 12.5 kbp and is polyadenylated at the 3' terminus of one strand. Molecular hybridization experiments indicate that there is sequence similarity between the NB58 dsRNA and dsRNAs from European isolates of C. parasitica, but not among dsRNAs of NB58 and those associated with other North American isolates. Hybridization experiments with mapped cDNA clones representing different regions of the 12.5 kbp dsRNA indicate that the termini and the 3'-proximal two-thirds (relative to the plus strand) are more conserved among NB58 and the European isolates than the rest of the 5'-proximal one-third. Nucleotide sequence analysis of the termini of NB58 dsRNA suggests common organizational features between it and the dsRNA from French-derived strain EP713. PMID:1545222

  5. Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.).

    PubMed

    Rodrigues, Simone M; Soares, Virgínia L F; de Oliveira, Tahise M; Gesteira, Abelmon S; Otoni, Wagner C; Costa, Marcio G C

    2007-11-01

    The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library. PMID:17952668

  6. Gold Nanoparticle Interference Study during the Isolation, Quantification, Purity and Integrity Analysis of RNA

    PubMed Central

    Sanabria, Natasha M.; Vetten, Melissa; Andraos, Charlene; Boodhia, Kailen; Gulumian, Mary

    2014-01-01

    Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220–340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190–220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190–220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects. PMID:25470814

  7. Isolation and Amplification of mRNA within a Simple Microfluidic Lab on a Chip

    PubMed Central

    Reinholt, Sarah J.; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J.

    2014-01-01

    The major modules for realizing molecular biological assays in a micro total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic messenger RNA (mRNA) within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid non-specific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 1015 fold and still result in successful on-chip re-amplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to bench-top devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes. PMID:24328414

  8. A comprehensive comparison of general RNA-RNA interaction prediction methods.

    PubMed

    Lai, Daniel; Meyer, Irmtraud M

    2016-04-20

    RNA-RNA interactions are fast emerging as a major functional component in many newly discovered non-coding RNAs. Basepairing is believed to be a major contributor to the stability of these intermolecular interactions, much like intramolecular basepairs formed in RNA secondary structure. As such, using algorithms similar to those for predicting RNA secondary structure, computational methods have been recently developed for the prediction of RNA-RNA interactions.We provide the first comprehensive comparison comprising 14 methods that predict general intermolecular basepairs. To evaluate these, we compile an extensive data set of 54 experimentally confirmed fungal snoRNA-rRNA interactions and 102 bacterial sRNA-mRNA interactions. We test the performance accuracy of all methods, evaluating the effects of tool settings, sequence length, and multiple sequence alignment usage and quality.Our results show that-unlike for RNA secondary structure prediction-the overall best performing tools are non-comparative energy-based tools utilizing accessibility information that predict short interactions on this data set. Furthermore, we find that maintaining high accuracy across biologically different data sets and increasing input lengths remains a huge challenge, causing implications forde novotranscriptome-wide searches. Finally, we make our interaction data set publicly available for future development and benchmarking efforts. PMID:26673718

  9. New Site of Modification of 23S rRNA Associated with Clarithromycin Resistance of Helicobacter pylori Clinical Isolates

    PubMed Central

    Fontana, Carla; Favaro, Marco; Minelli, Silvia; Criscuolo, Anna Angela; Pietroiusti, Antonio; Galante, Alberto; Favalli, Cartesio

    2002-01-01

    Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 μg/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates. PMID:12435674

  10. Expression of lipoprotein lipase mRNA and secretion in macrophages isolated from human atherosclerotic aorta.

    PubMed

    Mattsson, L; Johansson, H; Ottosson, M; Bondjers, G; Wiklund, O

    1993-10-01

    The expression of lipoprotein lipase (LPL) mRNA and the LPL activity were studied in macrophages (CD14 positive) from human atherosclerotic tissue. Macrophages were isolated after collagenase digestion by immunomagnetic isolation. About 90% of the cells were foam cells with oil red O positive lipid droplets. To analyze the mRNA expression, PCR with specific primers for LPL was used. Arterial macrophages were analyzed directly after isolation and the data showed low expression of LPL mRNA when compared with monocyte-derived macrophages. To induce the expression of LPL mRNA in macrophages, PMA was used. When incubating arterial macrophages with PMA for 24 h we could not detect any increase in LPL mRNA levels. Similarly, the cells secreted very small amounts of LPL even after PMA stimulation. In conclusion, these studies show a very low expression of LPL mRNA in the CD14-positive macrophage-derived foam cells isolated from human atherosclerotic tissue. These data suggest that the CD14-positive cells are a subpopulation of foam cells that express low levels of lipoprotein lipase, and the lipid content could be a major factor for downregulation of LPL. However, the cells were isolated from advanced atherosclerotic lesions, and these findings may not reflect the situation in early fatty streaks. PMID:8408628

  11. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter

    PubMed Central

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  12. End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter.

    PubMed

    Ide, Satoru; Dejardin, Jerome

    2015-01-01

    The unbiased identification of proteins associated with specific loci is crucial for understanding chromatin-based processes. The proteomics of isolated chromatin fragment (PICh) method has previously been developed to purify telomeres and identify associated proteins. This approach is based on the affinity capture of endogenous chromatin segments by hybridization with oligonucleotide containing locked nucleic acids. However, PICh is only efficient with highly abundant genomic targets, limiting its applicability. Here we develop an approach for identifying factors bound to the promoter region of the ribosomal RNA genes that we call end-targeting PICh (ePICh). Using ePICh, we could specifically enrich the RNA polymerase I pre-initiation complex, including the selectivity factor 1. The high purity of the ePICh material allowed the identification of ZFP106, a novel factor regulating transcription initiation by targeting RNA polymerase I to the promoter. Our results demonstrate that ePICh can uncover novel proteins controlling endogenous regulatory elements in mammals. PMID:25812914

  13. Isolation of genes involved in colorectal metastasis by differential display of mRNA species

    SciTech Connect

    Gustafson, C.; Chenevix-Trench, G.; Antalis, T.

    1994-09-01

    The genetic events that give rise to malignant colorectal tumors have been determined in some detail. Much less is known about the genes involved in metastasis of these neoplasms. A useful resource to study this process is the pair of cell lines, SW480 and SW620, which are derived from the primary and metastatic components, respectively, of the same colorectal tumor. We are using the method of differential display of mRNA species to isolate genes that are differentially expressed in these two cell lines. Differential display is carried out in triplicate, using three different RNA extractions from each cell line. Only fragments that are consistently up- or down-regulated in one cell line compared to another are examined further. Less than 1% of fragments are differentially expressed. These are cloned, sequenced, and used for Northern blot and reverse transcriptase-PCR in order to examine their differential expression further. The RNA sources for this expression analysis are (i) SW480 and SW620 cells, (ii) other pairs of primary and metastatic colorectal cell lines, (iii) primary and metastatic tissue from patients with colorectal cancer.

  14. rRNA-based method for sensitive detection of Babesia bigemina in bovine blood.

    PubMed Central

    Reddy, G R; Dame, J B

    1992-01-01

    Three synthetic oligonucleotide probes complementary to unique regions of Babesia bigemina small-subunit rRNA were developed for detecting the parasite in bovine blood. These probes specifically detected a parasitemia of 2 x 10(-5)% by autoradiography in less than 24 h by using a 200-microliters sample of bovine blood. These probes did not bind to total RNA or genomic DNA isolated from another closely related species, Babesia bovis, or to bovine leukocyte RNA. This method detected B. bigemina infections in calves inoculated with as few as 1,000 infected erythrocytes from the second day onward for 16 days. Images PMID:1629339

  15. Polymorphism of viral dsRNA in Xanthophyllomyces dendrorhous strains isolated from different geographic areas

    PubMed Central

    Baeza, Marcelo; Sanhueza, Mario; Flores, Oriana; Oviedo, Vicente; Libkind, Diego; Cifuentes, Víctor

    2009-01-01

    Background Strains of the astaxanthin producing yeast Xanthophyllomyces dendrorhous have been isolated from different cold regions around the earth, and the presence of double stranded RNA (dsRNA) elements was described in some isolates. This kind of viruses is widely distributed among yeasts and filamentous fungi and, although generally are cryptic in function, their studies have been a key factor in the knowledge of important fungi. In this work, the characterization and genetic relationships among dsRNA elements were determined in strains representatives of almost all regions of the earth where X. dendrorhous have been isolated. Results Almost all strains of X. dendrorhous analyzed carry one, two or four dsRNA elements, of molecular sizes in the range from 0.8 to 5.0 kb. Different dsRNA-patterns were observed in strains with different geographic origin, being L1 (5.0 kb) the common dsRNA element. By hybridization assays a high genomic polymorphism was observed among L1 dsRNAs of different X. dendrorhous strains. Contrary, hybridization was observed between L1 and L2 dsRNAs of strains from same or different regions, while the dsRNA elements of minor sizes (M, S1, and S2) present in several strains did not show hybridization with neither L1 or L2 dsRNAs. Along the growth curve of UCD 67-385 (harboring four dsRNAs) an increase of L2 relative to L1 dsRNA was observed, whiles the S1/L1 ratio remains constant, as well as the M/L1 ratio of Patagonian strain. Strains cured of S2 dsRNA were obtained by treatment with anisomycin, and comparison of its dsRNA contents with uncured strain, revealed an increase of L1 dsRNA while the L2 and S1 dsRNA remain unaltered. Conclusion The dsRNA elements of X. dendrorhous are highly variable in size and sequence, and the dsRNA pattern is specific to the geographic region of isolation. Each L1 and L2 dsRNA are viral elements able to self replicate and to coexist into a cell, and L1 and S2 dsRNAs elements could be part of a helper

  16. Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

    PubMed

    Nath, B Surendra; Gupta, S K; Bajpai, A K

    2012-12-01

    The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect. PMID:23129193

  17. [Antimicrobial susceptibilities of clinical Nocardia isolates identified by 16S rRNA gene sequence analysis].

    PubMed

    Uner, Mahmut Celalettin; Hasçelik, Gülşen; Müştak, Hamit Kaan

    2016-01-01

    Nocardia species are ubiquitous in the environment and responsible for various human infections such as pulmonary, cutaneous, central nervous system and disseminated nocardiosis. Since the clinical pictures and antimicrobial susceptibilities of Nocardia species exhibit variability, susceptibility testing is recommended for every Nocardia isolates. The aims of this study was to determine the antimicrobial susceptibilities of Nocardia clinical isolates and to compare the results of broth microdilution and disc diffusion susceptibility tests. A total of 45 clinical Nocardia isolates (isolated from 17 respiratory tract, 8 brain abscess, 7 pus, 3 skin, 3 conjunctiva, 2 blood, 2 tissue, 2 pleural fluid and 1 cerebrospinal fluid samples) were identified by using conventional methods and 16S rRNA gene sequence analysis. Susceptibility testing was performed for amikacin, ciprofloxacin, ceftriaxone, linezolid and trimethoprim-sulfamethoxazole (TMP-SMX) by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) criteria recommended in 2011 approved standard (M24-A2) and disk diffusion method used as an alternative comparative susceptibility testing method. Among the 45 Nocardia strains, N.cyriacigeorgica (n: 26, 57.8%) was the most common species, followed by N.farcinica (n: 12, 26.7%), N.otitiscaviarum (n: 4, 8.9%), N.asteroides (n: 1, 2.2%), N.neocaledoniensis (n: 1, 2.2%) and N.abscessus (n: 1, 2.2%). Amikacin and linezolid were the only two antimicrobials to which all isolates were susceptible for both broth microdilution and disk diffusion tests. In broth microdilution test, resistance rates to TMP-SMX, ceftriaxone and ciprofloxacin were found as 15.6%, 37.8% and 84.4% respectively, whereas in the disk diffusion test, the highest resistance rate was observed against ciprofloxacin (n: 33, 73.3%), followed by TMP-SMX (n: 22, 48.9%) and ceftriaxone (n: 15, 33.3%). In both of these tests, N.cyriacigeorgica was the species with the

  18. Computational Methods for RNA Structure Validation and Improvement.

    PubMed

    Jain, Swati; Richardson, David C; Richardson, Jane S

    2015-01-01

    With increasing recognition of the roles RNA molecules and RNA/protein complexes play in an unexpected variety of biological processes, understanding of RNA structure-function relationships is of high current importance. To make clean biological interpretations from three-dimensional structures, it is imperative to have high-quality, accurate RNA crystal structures available, and the community has thoroughly embraced that goal. However, due to the many degrees of freedom inherent in RNA structure (especially for the backbone), it is a significant challenge to succeed in building accurate experimental models for RNA structures. This chapter describes the tools and techniques our research group and our collaborators have developed over the years to help RNA structural biologists both evaluate and achieve better accuracy. Expert analysis of large, high-resolution, quality-conscious RNA datasets provides the fundamental information that enables automated methods for robust and efficient error diagnosis in validating RNA structures at all resolutions. The even more crucial goal of correcting the diagnosed outliers has steadily developed toward highly effective, computationally based techniques. Automation enables solving complex issues in large RNA structures, but cannot circumvent the need for thoughtful examination of local details, and so we also provide some guidance for interpreting and acting on the results of current structure validation for RNA. PMID:26068742

  19. A Raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new Raspberry bushy dwarf virus isolate was found in commercial blackberry (Rubus glaucus) in Azuay, province of Ecuador and named RBDV-Ec-Az. The complete bipartite genome was sequenced using dsRNA as initial template. RNA 1 was 5449 nucleotides (nt) long and the normal RBDV RNA 2 was 2231 nt lon...

  20. Immunochemical isolation of gamma-globulin mRNA and estimation of immunoglobulin gene reiteration.

    PubMed

    Muto, M

    1977-01-01

    The polyribosomes synthesizing gamma-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific gamma-globulin-synthesizing activity (cpm of gamma-globulin synthesized/microgram RNA) of this RNA was 10-fold greater than that from total polysomes. gamma-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis. This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the "half Cot value" is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains. PMID:927224

  1. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    PubMed Central

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  2. A simple and efficient method for isolating small RNAs from different plant species

    PubMed Central

    2011-01-01

    Background Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species. Results We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays. Conclusion Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays. PMID:21349188

  3. Efficient prediction methods for selecting effective siRNA sequences.

    PubMed

    Takasaki, Shigeru

    2010-02-01

    Although short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells, its gene silencing efficacy varies markedly and there are only a few consistencies among the recently reported design rules/guidelines for selecting siRNA sequences effective for mammalian genes. Another shortcoming of the previously reported methods is that they cannot estimate the probability that a candidate sequence will silence the target gene. This paper first reviewed the recently reported siRNA design guidelines and clarified the problems concerning the guidelines. It then proposed two prediction methods-Radial Basis Function (RBF) network and decision tree learning-and their combined method for selecting effective siRNA target sequences from many possible candidate sequences. They are quite different from the previous score-based siRNA design techniques and can predict the probability that a candidate siRNA sequence will be effective. The methods imply high estimation accuracy for selecting candidate siRNA sequences. PMID:20022002

  4. Double-stranded RNA viral infection of Trichomonas vaginalis and correlation with genetic polymorphism of isolates.

    PubMed

    Fraga, Jorge; Rojas, Lazara; Sariego, Idalia; Fernández-Calienes, Ayme

    2011-02-01

    Trichomonas vaginalis can be infected with double-stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. The objective of this study was to determine the possible correlation between the T. vaginalis genetic polymorphism and the isolate infection with TVV. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 37 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with TVV. The trees drawn based on RAPD data showed significantly association with the presence of TVV (P = 0.028) demonstrating the existence of concordance between the genetic relatedness and the presence of TVV in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the viral enters and/or survival. PMID:20875411

  5. The RNA 3D Motif Atlas: Computational methods for extraction, organization and evaluation of RNA motifs.

    PubMed

    Parlea, Lorena G; Sweeney, Blake A; Hosseini-Asanjan, Maryam; Zirbel, Craig L; Leontis, Neocles B

    2016-07-01

    RNA 3D motifs occupy places in structured RNA molecules that correspond to the hairpin, internal and multi-helix junction "loops" of their secondary structure representations. As many as 40% of the nucleotides of an RNA molecule can belong to these structural elements, which are distinct from the regular double helical regions formed by contiguous AU, GC, and GU Watson-Crick basepairs. With the large number of atomic- or near atomic-resolution 3D structures appearing in a steady stream in the PDB/NDB structure databases, the automated identification, extraction, comparison, clustering and visualization of these structural elements presents an opportunity to enhance RNA science. Three broad applications are: (1) identification of modular, autonomous structural units for RNA nanotechnology, nanobiology and synthetic biology applications; (2) bioinformatic analysis to improve RNA 3D structure prediction from sequence; and (3) creation of searchable databases for exploring the binding specificities, structural flexibility, and dynamics of these RNA elements. In this contribution, we review methods developed for computational extraction of hairpin and internal loop motifs from a non-redundant set of high-quality RNA 3D structures. We provide a statistical summary of the extracted hairpin and internal loop motifs in the most recent version of the RNA 3D Motif Atlas. We also explore the reliability and accuracy of the extraction process by examining its performance in clustering recurrent motifs from homologous ribosomal RNA (rRNA) structures. We conclude with a summary of remaining challenges, especially with regard to extraction of multi-helix junction motifs. PMID:27125735

  6. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

    NASA Technical Reports Server (NTRS)

    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  7. Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells.

    PubMed Central

    Gilmour, R S; Allan, M; Paul, J

    1984-01-01

    The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6). Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose. A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts. It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo. Images PMID:6330734

  8. Variation in the Spacer Regions Separating tRNA Genes in Renibacterium salmoninarum Distinguishes Recent Clinical Isolates from the Same Location

    PubMed Central

    Alexander, Sarah M.; Grayson, T. Hilton; Chambers, Edel M.; Cooper, Lynne F.; Barker, Gavin A.; Gilpin, Martyn L.

    2001-01-01

    A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles. PMID:11136759

  9. Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location.

    PubMed

    Alexander, S M; Grayson, T H; Chambers, E M; Cooper, L F; Barker, G A; Gilpin, M L

    2001-01-01

    A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles. PMID:11136759

  10. 16SrRNA sequencing of Dye decolorizing bacteria isolated from Soil

    PubMed Central

    Kumar, Avnish; Asthana, Monika; Gupta, Poonam; Yadav, Shweta; Sharma, Deepti; Singh, Km Neeraj; Kumar, Sunil

    2015-01-01

    Dye׳s residues in textile effluents are hazardous for humans and animals health. Such pollutants can be degraded into non-harmful molecules using biological approaches that are considered cheaper and ecologically safer. Isolated 15 bacterial cultures from soil that could be used in biological system were showed decolorization capacity for Acid Green dye (33.9% to 94.0%) using thin layer chromatography and broth culture method. The most promising cultures (AMC3) to decolorize Acid green Dye (94.6%) was re-coded as NSDSUAM for submitting at IMTECH, Chandigarh for sequencing. The 16SrRNA sequencing suggested that it can be a variant of Pseudomonas geniculata (99.85% identical similarity) with difference of 2 base pairs to reference strain Pseudomonas geniculata ATCC 19374(T). Thus present study proposed dye decolorizing efficiency of the isolated strain of Pseudomonas geniculata that was previously unnoticed. The sequence is deposited in NCBI GenBank with the accession number KP238100. PMID:25780272

  11. Isolation of the protein and RNA content of active sites of transcription from mammalian cells.

    PubMed

    Melnik, Svitlana; Caudron-Herger, Maïwen; Brant, Lilija; Carr, Ian M; Rippe, Karsten; Cook, Peter R; Papantonis, Argyris

    2016-03-01

    Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I-mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription. PMID:26914315

  12. Method of vibration isolating an aircraft engine

    NASA Technical Reports Server (NTRS)

    Bender, Stanley I. (Inventor); Butler, Lawrence (Inventor); Dawes, Peter W. (Inventor)

    1991-01-01

    A method for coupling an engine to a support frame for mounting to a fuselage of an aircraft using a three point vibration isolating mounting system in which the load reactive forces at each mounting point are statically and dynamically determined. A first vibration isolating mount pivotably couples a first end of an elongated support beam to a stator portion of an engine with the pivoting action of the vibration mount being oriented such that it is pivotable about a line parallel to a center line of the engine. An aft end of the supporting frame is coupled to the engine through an additional pair of vibration isolating mounts with the mounts being oriented such that they are pivotable about a circumference of the engine. The aft mounts are symmetrically spaced to each side of the supporting frame by 45 degrees. The relative orientation between the front mount and the pair of rear mounts is such that only the rear mounts provide load reactive forces parallel to the engine center line, in support of the engine to the aircraft against thrust forces. The forward mount is oriented so as to provide only radial forces to the engine and some lifting forces to maintain the engine in position adjacent a fuselage. Since each mount is connected to provide specific forces to support the engine, forces required of each mount are statically and dynamically determinable.

  13. Comparison of ribosomal RNA removal methods for transcriptome sequencing workflows in teleost fish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA sequencing (RNA-Seq) is becoming the standard for transcriptome analysis. Removal of contaminating ribosomal RNA (rRNA) is a priority in the preparation of libraries suitable for sequencing. rRNAs are commonly removed from total RNA via either mRNA selection or rRNA depletion. These methods have...

  14. Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

    SciTech Connect

    He, Shaomei; Wurtzel, Omri; Singh, Kanwar; Froula, Jeff L; Yilmaz, Suzan; Tringe, Susannah G; Wang, Zhong; Chen, Feng; Lindquist, Erika A; Sorek, Rotem; Hugenholtz, Philip

    2010-10-01

    The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

  15. RVboost: RNA-seq variants prioritization using a boosting method

    PubMed Central

    Wang, Chen; Davila, Jaime I.; Baheti, Saurabh; Bhagwate, Aditya V.; Wang, Xue; Kocher, Jean-Pierre A.; Slager, Susan L.; Feldman, Andrew L.; Novak, Anne J.; Cerhan, James R.; Thompson, E. Aubrey; Asmann, Yan W.

    2014-01-01

    Motivation: RNA-seq has become the method of choice to quantify genes and exons, discover novel transcripts and detect fusion genes. However, reliable variant identification from RNA-seq data remains challenging because of the complexities of the transcriptome, the challenges of accurately mapping exon boundary spanning reads and the bias introduced during the sequencing library preparation. Method: We developed RVboost, a novel method specific for RNA variant prioritization. RVboost uses several attributes unique in the process of RNA library preparation, sequencing and RNA-seq data analyses. It uses a boosting method to train a model of ‘good quality’ variants using common variants from HapMap, and prioritizes and calls the RNA variants based on the trained model. We packaged RVboost in a comprehensive workflow, which integrates tools of variant calling, annotation and filtering. Results: RVboost consistently outperforms the variant quality score recalibration from the Genome Analysis Tool Kit and the RNA-seq variant-calling pipeline SNPiR in 12 RNA-seq samples using ground-truth variants from paired exome sequencing data. Several RNA-seq–specific attributes were identified as critical to differentiate true and false variants, including the distance of the variant positions to exon boundaries, and the percent of the reads supporting the variant in the first six base pairs. The latter identifies false variants introduced by the random hexamer priming during the library construction. Availability and implementation: The RVboost package is implemented to readily run in Mac or Linux environments. The software and user manual are available at http://bioinformaticstools.mayo.edu/research/rvboost/. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25170027

  16. Comparison between rpoB and 16S rRNA Gene Sequencing for Molecular Identification of 168 Clinical Isolates of Corynebacterium

    PubMed Central

    Khamis, Atieh; Raoult, Didier; La Scola, Bernard

    2005-01-01

    Higher proportions (91%) of 168 corynebacterial isolates were positively identified by partial rpoB gene determination than by that based on 16S rRNA gene sequences. This method is thus a simple, molecular-analysis-based method for identification of corynebacteria, but it should be used in conjunction with other tests for definitive identification. PMID:15815024

  17. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

    PubMed Central

    Laurent, Louise C.; Abdel-Mageed, Asim B.; Adelson, P. David; Arango, Jorge; Balaj, Leonora; Breakefield, Xandra; Carlson, Elizabeth; Carter, Bob S.; Majem, Blanca; Chen, Clark C.; Cocucci, Emanuele; Danielson, Kirsty; Courtright, Amanda; Das, Saumya; Elmageed, Zakaria Y. Abd; Enderle, Daniel; Ezrin, Alan; Ferrer, Marc; Freedman, Jane; Galas, David; Gandhi, Roopali; Huentelman, Matthew J.; Van Keuren-Jensen, Kendall; Kalani, Yashar; Kim, Yong; Krichevsky, Anna M.; Lai, Charles; Lal-Nag, Madhu; Laurent, Clara D.; Leonardo, Trevor; Li, Feng; Malenica, Ivana; Mondal, Debasis; Nejad, Parham; Patel, Tushar; Raffai, Robert L.; Rubio, Renee; Skog, Johan; Spetzler, Robert; Sun, Jie; Tanriverdi, Kahraman; Vickers, Kasey; Wang, Liang; Wang, Yaoyu; Wei, Zhiyun; Weiner, Howard L.; Wong, David; Yan, Irene K.; Yeri, Ashish; Gould, Stephen

    2015-01-01

    Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field. PMID:26320937

  18. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

    PubMed Central

    Hartmann, R K; Erdmann, V A

    1989-01-01

    A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA. Images PMID:2722737

  19. Methods and compositions for the production of orthogonal tRNA-aminoacyl-tRNA synthetase pairs

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason W.; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2011-09-06

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  20. Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Steven William; Zhang, Zhiwen

    2012-05-22

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  1. Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Steven William; Zhang, Zhiwen

    2008-04-08

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  2. Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2010-05-11

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  3. RNA-Skim: a rapid method for RNA-Seq quantification at transcript level

    PubMed Central

    Zhang, Zhaojun; Wang, Wei

    2014-01-01

    Motivation: RNA-Seq technique has been demonstrated as a revolutionary means for exploring transcriptome because it provides deep coverage and base pair-level resolution. RNA-Seq quantification is proven to be an efficient alternative to Microarray technique in gene expression study, and it is a critical component in RNA-Seq differential expression analysis. Most existing RNA-Seq quantification tools require the alignments of fragments to either a genome or a transcriptome, entailing a time-consuming and intricate alignment step. To improve the performance of RNA-Seq quantification, an alignment-free method, Sailfish, has been recently proposed to quantify transcript abundances using all k-mers in the transcriptome, demonstrating the feasibility of designing an efficient alignment-free method for transcriptome quantification. Even though Sailfish is substantially faster than alternative alignment-dependent methods such as Cufflinks, using all k-mers in the transcriptome quantification impedes the scalability of the method. Results: We propose a novel RNA-Seq quantification method, RNA-Skim, which partitions the transcriptome into disjoint transcript clusters based on sequence similarity, and introduces the notion of sig-mers, which are a special type of k-mers uniquely associated with each cluster. We demonstrate that the sig-mer counts within a cluster are sufficient for estimating transcript abundances with accuracy comparable with any state-of-the-art method. This enables RNA-Skim to perform transcript quantification on each cluster independently, reducing a complex optimization problem into smaller optimization tasks that can be run in parallel. As a result, RNA-Skim uses <4% of the k-mers and <10% of the CPU time required by Sailfish. It is able to finish transcriptome quantification in <10 min per sample by using just a single thread on a commodity computer, which represents >100 speedup over the state-of-the-art alignment-based methods, while delivering

  4. mRAP, a sensitive method for determination of microRNA expression profiles.

    PubMed

    Mano, Hiroyuki; Takada, Shuji

    2007-10-01

    MicroRNAs (miRNAs) are noncoding RNA molecules of 21-24 nucleotides that regulate the expression of target genes in a posttranscriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling strategy, termed miRNA amplification profiling (mRAP), that relies on the use of a long, optimized 5' adaptor, the SMART (switching mechanism at the 5' end of RNA templates of reverse transcriptase) method, the polymerase chain reaction, and cDNA concatamerization after BanI digestion. This approach is highly sensitive, readily allowing the isolation of > 1 x 10(4) independent miRNA-derived cDNAs from < or = 1 x 10(4) cells. The mRAP method thus makes it possible to analyze miRNA expression profiles for small quantities of tissue or cells such as fresh clinical specimens. PMID:17889798

  5. A Comparison of Sperm RNA-Seq Methods

    PubMed Central

    Mao, Shihong; Sendler, Edward; Goodrich, Robert J.; Hauser, Russ; Krawetz, Stephen A.

    2015-01-01

    A significant challenge to the effective application of RNA-seq to the complete transcript analysis of low quantity and/or degraded samples is the amplification of minimal input RNA to enable sequencing library construction. Several strategies have been commercialized in order to facilitate this goal. However, each strategy has its own specific protocols and methodology, and each may introduce unique bias and in some cases show specific preference for a collection of sequences. Our wider investigation of human spermatozoal RNAs was able to reveal their complexity despite being generally characterized by low quantity and high fragmentation. In this study, four commercially available RNA-seq library amplification protocols for the preparation of low quantity/highly fragmented samples - SMARTer™ Ultra Low RNA (SU) for Illumina® Sequencing (Clontech Laboratories, Inc.), SeqPlex RNA Amplification (SP) (Sigma-Aldrich Co.), Ovation® RNA-Seq System V2 (OR), and Ovation® RNA-Seq FFPE System from NuGEN (FFPE) (NuGEN Technologies, Inc.) - were assessed using human sperm RNAs. Further investigation analyzed the effects on the end results of two different library preparation methods - Encore NGS Multiplex System I (Enc) and Ovation Ultralow Library Systems (UL) (NuGEN Technologies, Inc.) - that appeared best suited to this type of RNA, along with other potential confounding factors such as Formalin Fixed Paraffin Embedded (FFPE) preservation. Our results indicate that for each library preparation protocol, the differences in the initial amount of input RNA and choice of RNA purification step do not generate marked differences in terms of RNA profiling. However, substantial disparity is introduced by individual amplification methods prior to library construction. These significant differences may be caused by the different priming methods or amplification strategies used in each of the four different protocols examined. The observation of intrasample variation introduced by

  6. System and Method for Isolation of Samples

    NASA Technical Reports Server (NTRS)

    Zhang, Ye (Inventor); Wu, Honglu (Inventor)

    2014-01-01

    Systems and methods for isolating samples are provided. The system comprises a first membrane and a second membrane disposed within an enclosure. First and second reservoirs can also be disposed within the enclosure and adapted to contain one or more reagents therein. A first valve can be disposed within the enclosure and in fluid communication with the first reservoir, the second reservoir, or both. The first valve can also be in fluid communication with the first or second membranes or both. The first valve can be adapted to selectively regulate the flow of the reagents from the first reservoir, through at least one of the first and second membranes, and into the second reservoir.

  7. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  8. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Moyzis, Robert K.; Ratliff, Robert L.; Shera, E. Brooks; Stewart, Carleton C.

    1990-01-01

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

  9. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  10. The development of a method of suspension RNA-FISH for forensically relevant epithelial cells using LNA probes.

    PubMed

    Williams, Eletra; Lin, Meng-Han; Harbison, Sallyann; Fleming, Rachel

    2014-03-01

    Messenger RNA profiling is becoming a common method for body fluid identification in forensic science but there are disadvantages when cell mixtures are present from more than one individual. A method that could identify and separate such cell mixtures would simplify downstream analysis. To do this, we have developed a novel method of RNA suspension-fluorescent in situ hybridization (RNA S-FISH) using a locked nucleic acid (LNA) probe for the keratin 10 (KRT10) mRNA that is suitable as a potential marker for epithelial cells. As sample size may be restricted in forensic samples, this method has focused on minimizing cell loss whilst maintaining signal strength. Furthermore, we have shown that it is possible to obtain full DNA profiles from 150 RNA S-FISH labeled cells isolated using laser microdissection. PMID:24528586

  11. Improved method for simultaneous isolation of proteins and nucleic acids.

    PubMed

    Chey, Soroth; Claus, Claudia; Liebert, Uwe Gerd

    2011-04-01

    Guanidinium thiocyanate-phenol-chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol-bromochloropropane-water was used for precipitation of proteins from the phenol-ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample. PMID:21094121

  12. Differences in 23S ribosomal RNA mutations between wild-type and mutant macrolide-resistant Chlamydia trachomatis isolates

    PubMed Central

    JIANG, YONG; ZHU, HUI; YANG, LI-NA; LIU, YUAN-JUN; HOU, SHU-PING; QI, MAN-LI; LIU, QUAN-ZHONG

    2015-01-01

    The aim of the present study was to determine the in vitro susceptibility of wild-type and mutant clinical isolates of Chlamydia (C.) trachomatis strains to erythromycin, azithromycin and josamycin, and to identify the resistance-conferring 23S ribosomal (r)RNA mutations in the isolates. The wild-type resistant isolates were defined as those with minimum inhibitory concentration values above the tissue concentration of the antibiotic in the urogenital system. Furthermore, all resistant C. trachomatis isolates were exposed to sub-inhibitory concentrations of macrolides, and 13 resistant mutants were selected following serial passages. Among the 8 wild-type isolates that were resistant to erythromycin, 3 isolates had a mutation at T2611C in the 23S rRNA gene while the others did not show any 23S rRNA mutations. The selected mutant isolates showed a 4- to 16-fold reduction in in vitro sensitivities. With regard to the mutant strains, the T2611C mutation was found in 10 isolates, A2057G mutation in 6 isolates, and A2059G mutation in 1 isolate. Thus, the macrolide-resistant isolates of the wild-type strain had different mutations from those selected by exposure to sub-inhibitory concentrations of macrolides. Also, since 23S rRNA mutations were not identified in certain isolates, it was considered that other molecular mechanisms may also be responsible for the macrolide resistance of C. trachomatis. PMID:26622462

  13. Isolating Influenza RNA from Clinical Samples Using Microfluidic Oil-Water Interfaces

    PubMed Central

    Cui, Francis R.; Wang, Jingjing; Opal, Steven M.; Tripathi, Anubhav

    2016-01-01

    The effective and robust separation of biomolecules of interest from patient samples is an essential step in diagnostic applications. We present a platform for the fast extraction of nucleic acids from clinical specimens utilizing paramagnetic PMPs, an oil-water interface, a small permanent magnet and a microfluidic channel to separate and purify captured nucleic acids from lysate in less than one minute, circumventing the need for multiple washing steps and greatly simplifying and expediting the purification procedure. Our device was able to isolate influenza RNA from clinical nasopharyngeal swab samples with high efficiency when compared to the Ambion® MagMAXTM Viral RNA Isolation Kit, sufficiently separating nucleic acid analytes from PCR-inhibiting contaminants within the lysate while also critically maintaining high integrity of the viral genome. We find that this design has great potential for rapid, efficient and sensitive nucleic acid separation from patient sample. PMID:26886007

  14. Small RNA Detection by in Situ Hybridization Methods

    PubMed Central

    Urbanek, Martyna O.; Nawrocka, Anna U.; Krzyzosiak, Wlodzimierz J.

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  15. Small RNA Detection by in Situ Hybridization Methods.

    PubMed

    Urbanek, Martyna O; Nawrocka, Anna U; Krzyzosiak, Wlodzimierz J

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  16. A Rapid Method for Isolating Glandular Trichomes

    PubMed Central

    Yerger, Ellen H.; Grazzini, Richard A.; Hesk, David; Cox-Foster, Diana L.; Craig, Richard; Mumma, Ralph O.

    1992-01-01

    A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium ×hortorum L.H. Bailey. These purified cells contained high concentrations of unusual ω5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no ω5-fatty acids, thereby unequivocally localizing ω5-fatty acids to the trichomes. Because ω5-fatty acids are unique precursors for the biosynthesis of ω5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes. ImagesFigure 1Figure 2 PMID:16668834

  17. Inverse PCR-based method for isolating novel SINEs from genome.

    PubMed

    Han, Yawei; Chen, Liping; Guan, Lihong; He, Shunping

    2014-04-01

    Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization. PMID:24122282

  18. Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources.

    PubMed

    Chomczynski, P; Mackey, K

    1995-12-01

    A modification of the TRI Reagent procedure has been elaborated for isolation of RNA from polysaccharide- and proteoglycan-rich material. In the modified procedure, RNA is precipitated from the aqueous phase by the combined action of isopropanol and a high-salt concentration. Under these conditions, RNA is effectively precipitated while contaminating polysaccharides and proteoglycans remain in the soluble form. The modified precipitation does not prolong or increase the complexity of the TRI Reagent procedure. The new procedure was tested by isolation of RNA from polysaccharide- and proteoglycan-rich tissues such as rat liver and aorta. PMID:8747660

  19. A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.

    PubMed

    Ziros, Panos G; Chartoumpekis, Dionysios V; Sykiotis, Gerasimos P

    2016-01-01

    As a dedicated hormone-secreting organ, the thyroid gland possesses a complement of proteostatic systems, including antioxidant, unfolded protein, and autophagic responses. The vast majority of animal investigations of thyroid physiology and, more recently, proteostasis, have utilized as model the rat, rather than the mouse. This is due to the very small size of the thyroid gland in the latter, with a total weight of ~2 mg (~1 mg per thyroid lobe). However, this strategy has limited the utilization of genetic approaches, such as taking advantage of the various transgenic and knockout mouse models. Here, we describe a simple and highly efficient protocol for the simultaneous isolation of mRNA, micro-RNA and 150-200 μg of protein from as little as 1 mg of mouse thyroid tissue, the average weight of one of the two thyroid lobes, thus preserving the other lobe for immunohistochemical or other analyses. While our workflow is similar to other protocols published in the literature and/or proposed by commercial reagent providers, we have introduced a key modification that addresses efficiently the most challenging step of the protein isolation process: the solubilization of the protein pellet after RNA extraction and protein precipitation. We demonstrate the feasibility of our approach and its utility for downstream analyses (including Western blotting) that facilitate the comparative study of proteostatic pathways in the mouse thyroid. We have also successfully applied this protocol on samples from mouse liver, brown and white adipose tissue, as well as from rodent cell lines. PMID:27613051

  20. An efficient method for long-term room temperature storage of RNA

    PubMed Central

    Fabre, Anne-Lise; Colotte, Marthe; Luis, Aurélie; Tuffet, Sophie; Bonnet, Jacques

    2014-01-01

    RNA is a tool used in many fields, from molecular and cellular biology to medicine and nanotechnology. For most of these uses, the integrity of RNA is required and must be maintained during storage. Even though freezing is currently the storage method of choice, the increasing number of samples to be stored and the costly use of a cold chain have highlighted the need for room temperature preservation methods. Here, we report a new room temperature technology that consists in drying RNA samples in the presence of a stabilizer in stainless steel minicapsules. These air- and water-tight capsules isolate RNA from the atmosphere and maintain an anhydrous and anoxic environment. Through the evaluation of RNA integrity over time at room temperature or 90 °C, we identified atmospheric humidity as a major deleterious factor. The degradation rate dependence in temperature fitted an Arrhenius model, with an activation energy of 28.5 kcal/mol and an extrapolated room temperature degradation rate of 3.2 10−13/nt/s (95% confidence interval: 2.3–4.2/nt/s). In these conditions, it is expected that an RNA molecule will be subjected to 0.7–1.3 cut every 1000 nucleotides per century. In addition, we showed that stored RNA is compatible for further analyses, such as reverse transcription-quantitative PCR. No significant change in the Cq values was observed over a simulated period of several decades. At last, our data are consistent with a sequence-independent degradation rate of RNA in the solid state. PMID:23860045

  1. Method for Confirming Cytoplasmic Delivery of RNA Aptamers

    PubMed Central

    Dickey, David D; Dassie, Justin P; Giangrande, Paloma H

    2016-01-01

    RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity. PMID:26472453

  2. Emergence of Multidrug-Resistant Campylobacter Species Isolates with a Horizontally Acquired rRNA Methylase

    PubMed Central

    Wang, Yang; Zhang, Maojun; Deng, Fengru; Shen, Zhangqi; Wu, Congming; Zhang, Jianzhong

    2014-01-01

    Antibiotic-resistant Campylobacter constitutes a serious threat to public health, and resistance to macrolides is of particular concern, as this class of antibiotics is the drug of choice for clinical therapy of campylobacteriosis. Very recently, a horizontally transferrable macrolide resistance mediated by the rRNA methylase gene erm(B) was reported in a Campylobacter coli isolate, but little is known about the dissemination of erm(B) among Campylobacter isolates and the association of erm(B)-carrying isolates with clinical disease. To address this question and facilitate the control of antibiotic-resistant Campylobacter, we determined the distribution of erm(B) in 1,554 C. coli and Campylobacter jejuni isolates derived from food-producing animals and clinically confirmed human diarrheal cases. The results revealed that 58 of the examined isolates harbored erm(B) and exhibited high-level resistance to macrolides, and most were recent isolates, derived in 2011-2012. In addition, the erm(B)-positive isolates were all resistant to fluoroquinolones, another clinically important antibiotic used for treating campylobacteriosis. The erm(B) gene is found to be associated with chromosomal multidrug resistance genomic islands (MDRGIs) of Gram-positive origin or with plasmids of various sizes. All MDRGIs were transferrable to macrolide-susceptible C. jejuni by natural transformation under laboratory conditions. Molecular typing of the erm(B)-carrying isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) identified diverse genotypes and outbreak-associated diarrheal isolates. Molecular typing also suggested zoonotic transmission of erm(B)-positive Campylobacter. These findings reveal an emerging and alarming trend of dissemination of erm(B) and MDRGIs in Campylobacter and underscore the need for heightened efforts to control their further spread. PMID:24982085

  3. A rapid method for assessing the RNA-binding potential of a protein

    PubMed Central

    Bendak, K.; Loughlin, F.E.; Cheung, V.; O’Connell, M.R.; Crossley, M.; Mackay, J.P.

    2012-01-01

    In recent years, evidence has emerged for the existence of many diverse types of RNA, which play roles in a wide range of biological processes in all kingdoms of life. These molecules generally do not, however, act in isolation, and identifying which proteins partner with RNA is a major challenge. Many methods, in vivo and in vitro, have been used to address this question, including combinatorial or high-throughput approaches, such as systematic evolution of ligands, cross-linking and immunoprecipitation and RNA immunoprecipitation combined with deep sequencing. However, most of these methods are not trivial to pursue and often require substantial optimization before results can be achieved. Here, we demonstrate a simple technique that allows one to screen proteins for RNA-binding properties in a gel-shift experiment and can be easily implemented in any laboratory. This assay should be a useful first-pass tool for assessing whether a protein has RNA- or DNA-binding properties, prior to committing resources to more complex procedures. PMID:22492509

  4. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation

    PubMed Central

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M.; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-01-01

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice. PMID:26216949

  5. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation.

    PubMed

    Chen, Liang; Rashid, Farooq; Shah, Abdullah; Awan, Hassaan M; Wu, Mingming; Liu, An; Wang, Jun; Zhu, Tao; Luo, Zhaofeng; Shan, Ge

    2015-08-11

    p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice. PMID:26216949

  6. Drop-on-Demand Single Cell Isolation and Total RNA Analysis

    PubMed Central

    Moon, Sangjun; Kim, Yun-Gon; Dong, Lingsheng; Lombardi, Michael; Haeggstrom, Edward; Jensen, Roderick V.; Hsiao, Li-Li; Demirci, Utkan

    2011-01-01

    Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods. PMID:21412416

  7. Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

    PubMed Central

    Atkins, Norman; Miller, Charlie M.; Owens, Joseph R.; Turek, Fred W.

    2011-01-01

    As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies. Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-μm serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection

  8. Isolation and characterization of the gene coding for Escherichia coli arginyl-tRNA synthetase.

    PubMed Central

    Eriani, G; Dirheimer, G; Gangloff, J

    1989-01-01

    The gene coding for Escherichia coli arginyl-tRNA synthetase (argS) was isolated as a fragment of 2.4 kb after analysis and subcloning of recombinant plasmids from the Clarke and Carbon library. The clone bearing the gene overproduces arginyl-tRNA synthetase by a factor 100. This means that the enzyme represents more than 20% of the cellular total protein content. Sequencing revealed that the fragment contains a unique open reading frame of 1734 bp flanked at its 5' and 3' ends respectively by 247 bp and 397 bp. The length of the corresponding protein (577 aa) is well consistent with earlier Mr determination (about 70 kd). Primer extension analysis of the ArgRS mRNA by reverse transcriptase, located its 5' end respectively at 8 and 30 nucleotides downstream of a TATA and a TTGAC like element (CTGAC) and 60 nucleotides upstream of the unusual translation initiation codon GUG; nuclease S1 analysis located the 3'-end at 48 bp downstream of the translation termination codon. argS has a codon usage pattern typical for highly expressed E. coli genes. With the exception of the presence of a HVGH sequence similar to the HIGH consensus element, ArgRS has no relevant sequence homologies with other aminoacyl-tRNA synthetases. Images PMID:2668891

  9. Isolation and in vitro translation of mRNA from rat peritoneal mast cells and rat basophilic leukemia cells.

    PubMed

    Fujimaki, H; Lee, T D; Swieter, M; Saito, A; Tamaoki, T; Befus, A D

    1988-11-10

    In the absence of any specific literature on the isolation of RNA from mast cells, our initial attempts established that unusual measures would be needed to prepare acceptable yields of high quality RNA from peritoneal mast cells of normal adult rats. Accordingly, we developed procedures for the isolation and characterization of RNA from rat peritoneal mast cells (PMC) and basophilic leukemia cells (RBL). The significant components of the procedures include: separation and removal of mast cell granules to minimize contamination of RNA with proteins and proteoglycans; use of bentonite in phenol extractions; and repetition of extractions and precipitation. The amounts of total RNA extracted from PMC were about 15% of those from RBL, although the percentage mRNA of total RNA in PMC and RBL was similar (1.8 and 2.0%). Ribosomal RNA banding patterns in agarose gel electrophoresis and in vitro translation experiments indicate that the isolated RNA can be employed for analysis of molecular mechanisms of mast cell function and heterogeneity. PMID:3183393

  10. Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds

    PubMed Central

    2013-01-01

    Background High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants. Findings Here we report the use of adsorption property of activated charcoal (0.03%–0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number. Conclusions The RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds. PMID:23537338

  11. Defective minor spliceosome mRNA processing results in isolated familial growth hormone deficiency

    PubMed Central

    Argente, Jesús; Flores, Raquel; Gutiérrez-Arumí, Armand; Verma, Bhupendra; Martos-Moreno, Gabriel Á; Cuscó, Ivon; Oghabian, Ali; Chowen, Julie A; Frilander, Mikko J; Pérez-Jurado, Luis A

    2014-01-01

    The molecular basis of a significant number of cases of isolated growth hormone deficiency remains unknown. We describe three sisters affected with severe isolated growth hormone deficiency and pituitary hypoplasia caused by biallelic mutations in the RNPC3 gene, which codes for a minor spliceosome protein required for U11/U12 small nuclear ribonucleoprotein (snRNP) formation and splicing of U12-type introns. We found anomalies in U11/U12 di-snRNP formation and in splicing of multiple U12-type introns in patient cells. Defective transcripts include preprohormone convertases SPCS2 and SPCS3 and actin-related ARPC5L genes, which are candidates for the somatotroph-restricted dysfunction. The reported novel mechanism for familial growth hormone deficiency demonstrates that general mRNA processing defects of the minor spliceosome can lead to very narrow tissue-specific consequences. Subject Categories Genetics, Gene Therapy ' Genetic Disease; Metabolism PMID:24480542

  12. Isolation and characterization of the heterogeneous nuclear RNA-ribonucleoprotein complex

    SciTech Connect

    Choi, Y.D.

    1985-01-01

    Exposure of cells to UV light of sufficient intensity brings about crosslinking of RNA to proteins which are in direct contact with it in vivo. The major (/sup 35/S)methionine-labeled proteins which become crosslinked to poly(A)/sup +/hnRNA in HeLa cells are of 120K, 68K, 53K, 43K, 41K, 38K, and 36K (K = kilodaltons). By immunizing mice with UV crosslinked complexes two monoclonal antibodies (2B12 and 4F4) against the C proteins (41K and 43K) and one (3G6) against the 120K protein of the hnRNP complex were obtained. Immunofluorescence microscopy demonstrates that the C proteins and 120K are segregated to the nucleus and are not associated with nucleoli or chromatin. The two C proteins are highly related to each other antigenically. Monoclonal antibody 4F4 identifies the C proteins of the hnRNP complex in widely divergent species from human to lizard. The C proteins are phosphorylated and are in contact with hnRNA in vivo. The hnRNP complex was isolated from vertebrate cell nuclei by immunoprecipitation with these monoclonal antibodies. This complex contains proteins and hnRNA of up to approx.10 kb. The major steady state labeled (/sup 35/S)methionine labeled proteins of the isolated complex from HeLa cells are of 34K, 36K, 36K (A1 and A2), 37K, 38K (B1 and B2), 41K, 43K (C1 and C2) and doublets at 68K and at 120K. These proteins are organized into a 30S particle. Large hnRNP complexes are composed of multiples of 30S particles which are connected by highly nuclease sensitive stretches of hnRNA. It it concluded that the hnRNP structure is an integral component of the mRNA formation pathway in the eukaryotic cell.

  13. RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and mercury-labeled CTP.

    PubMed Central

    Ganguly, R; Banerjee, M R

    1978-01-01

    Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA. PMID:724523

  14. Comparison of functional limbal epithelial stem cell isolation methods.

    PubMed

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Dziasko, Marc; Galindo, Sara; Rey, Esther; Herreras, José M; Corrales, Rosa M; Daniels, Julie T; Calonge, Margarita

    2016-05-01

    The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either

  15. Application of an RNA amplification method for reliable single-cell transcriptome analysis

    PubMed Central

    Suslov, Oleg; Silver, Daniel J.; Siebzehnrubl, Florian A.; Orjalo, Arturo; Ptitsyn, Andrey; Steindler, Dennis A.

    2016-01-01

    Diverse cell types have unique transcriptional signatures that are best interrogated at single-cell resolution. Here we describe a novel RNA amplification approach that allows for high fidelity gene profiling of individual cells. This technique significantly diminishes the problem of 3′ bias, enabling detection of all regions of transcripts, including the recognition of mRNA with short or completely absent poly(A) tails, identification of noncoding RNAs, and discovery of the full array of splice isoforms from any given gene product. We assess this technique using statistical and bioinformatics analyses of microarray data to establish the limitations of the method. To demonstrate applicability, we profiled individual cells isolated from the mouse subventricular zone (SVZ)—a well-characterized, discrete yet highly heterogeneous neural structure involved in persistent neurogenesis. Importantly, this method revealed multiple splice variants of key germinal zone gene products within individual cells, as well as an unexpected coexpression of several mRNAs considered markers of distinct and separate SVZ cell types. These findings were independently confirmed using RNA-fluorescence in situ hybridization (RNA-FISH), contributing to the utility of this new technology that offers genomic and transcriptomic analysis of small numbers of dynamic and clinically relevant cells. PMID:26345506

  16. Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

    PubMed

    Huber, R; Burggraf, S; Mayer, T; Barns, S M; Rossnagel, P; Stetter, K O

    1995-07-01

    A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities. PMID:7541115

  17. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  18. The complete S RNA and M RNA nucleotide sequences of a hippeastrum chlorotic ringspot virus (HCRV) isolate from Hymenocallis littoralis (Jacq.) Salisb in China.

    PubMed

    Xu, Ye; Lou, Shi-gao; Li, Xiao-lin; Zheng, Yuan-xian; Wang, Wen-cheng; Liu, Ya-ting

    2013-12-01

    An isolate of hippeastrum chlorotic ringspot virus (HCRV) named HLS1-2, causing necrotic spots on leaves of spider lily, was obtained and characterized in China. The complete S RNA and M RNAs of the HLS1-2 isolate are 2724 nt and 4741 nt in length, respectively. The HLS1-2 S RNA sequence is most closely related to that of HCRV, with 99 % identity. Viral proteins encoded by the M RNA are closely related to those of tomato yellow ring virus (TYRV), polygonum ringspot virus (PolRSV) and iris yellow spot virus (IYSV). Phylogenetic trees for the four viral proteins encoded by the S and M RNAs placed HCRV-HLS1-2 in a distinct cluster with IYSV, TYRV and PolRSV and provided further support for the subdivision of tospoviruses into American and Eurasian groups. PMID:23812614

  19. Identification of coagulase-negative staphylococci isolated from ovine milk samples by PCR-RFLP of 16S rRNA and gap genes.

    PubMed

    Onni, T; Sanna, G; Cubeddu, G P; Marogna, G; Lollai, S; Leori, G; Tola, S

    2010-08-26

    The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR-RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR-RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR-RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis. PMID:20167442

  20. Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections

    PubMed Central

    Park, Joo Youn; Fox, Lawrence K.; Seo, Keun Seok; McGuire, Mark A.; Park, Yong Ho; Rurangirwa, Fred R.; Sischo, William M.; Bohach, Gregory A.

    2013-01-01

    Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0 % (200 / 263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0 % (255 / 263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system. PMID:20667671

  1. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    PubMed

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses. PMID:24078111

  2. The complete sequence of the genomic RNA of an isolate of Lily virus X (genus Potexvirus).

    PubMed

    Chen, J; Shi, Y-H; Adams, M J; Chen, J-P

    2005-04-01

    The complete sequence of the genomic RNA of an isolate of Lily virus X (LVX) has been determined for the first time. The isolate from the Netherlands was 5823 nucleotide (nt) long excluding the 3'-poly(A) tail, making it the shortest reported potexvirus sequence. The 5'-non-coding region begins with GGAAAA like that of Scallion virus X (ScaVX) and some isolates of Cymbidium mosaic virus (CymMV), whereas those of other sequenced potexviruses probably all begin with GAAAA. The genome organisation was similar to that of other members of the genus except that a TGBp3-like region lacked a normal AUG start codon. A phylogenetic analysis based on the entire coding sequence showed that LVX was most closely related to Strawberry mild yellow edge virus and belonged in a subgroup of the genus that also contains CymMV, Narcissus mosaic virus, ScaVX, Pepino mosaic virus, Potato aucuba mosaic virus and White clover mosaic virus. PMID:15578239

  3. NDM-1 Metallo-β-Lactamase and ArmA 16S rRNA methylase producing Providencia rettgeri clinical isolates in Nepal

    PubMed Central

    2014-01-01

    Background Drug-resistant Providencia rettgeri producing metallo-β-lactamase and 16S rRNA methylase has been reported in several countries. We analyzed P. rettgeri clinical isolates with resistance to carbapenems and aminoglycosides in a hospital in Nepal. Methods Five clinical isolates of multidrug-resistant P. rettgeri were obtained in a hospital in Nepal. Antimicrobial susceptibilities were determined using the microdilution method and entire genomes were sequenced to determine drug-resistant genes. Epidemiological analysis was performed by pulsed-field gel electrophoresis. Results Four of the 5 isolates were resistant to carbapenems (imipenem and meropenem), with MICs ≥16 mg/L, with the remaining isolate showing intermediate resistance to imipenem, with an MIC of 2 mg/L and susceptibility to meropenem with an MIC ≤1 mg/L. All 5 isolates had blaVEB-1. Of the 4 carbapenem-resistant strains, 3 had blaNDM-1 and 1 had blaOXA-72. All isolates were highly resistant to aminoglycosides (MICs ≥1,024 mg/L) and harbored armA. As the result of pulsed-field gel electrophoresis pattern analysis in the 5 P. rettgeri isolates, 4 had identical PFGE patterns and the fifth showed 95.7% similarity. Conclusions This is the first report describing multidrug-resistant P. rettgeri strains harboring blaNDM-1 or blaOXA-72 and armA isolated from patients in Nepal. PMID:24484534

  4. Method for nucleic acid isolation using supercritical fluids

    DOEpatents

    Nivens, D.E.; Applegate, B.M.

    1999-07-13

    A method is disclosed for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification. 4 figs.

  5. Method for nucleic acid isolation using supercritical fluids

    DOEpatents

    Nivens, David E.; Applegate, Bruce M.

    1999-01-01

    A method for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification.

  6. Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

    PubMed Central

    Hernandez, Luiza I.; Flenker, Katie S.; Hernandez, Frank J.; Klingelhutz, Aloysius J.; II, James O. McNamara; Giangrande, Paloma H.

    2013-01-01

    Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo. PMID:23894227

  7. Identification of Atypical Rhodococcus-Like Clinical Isolates as Dietzia spp. by 16S rRNA Gene Sequencing▿

    PubMed Central

    Pilares, Lilian; Agüero, Jesús; Vázquez-Boland, José A.; Martínez-Martínez, Luis; Navas, Jesús

    2010-01-01

    Rhodococcus equi and Dietzia spp. are closely related actinomycetes that show similar phenotypic properties. In humans, R. equi is an opportunistic pathogen associated with severe immunodeficiency. Dietzia spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as R. equi by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true R. equi strains, whereas the other eight were identified as members of the genus Dietzia, including Dietzia maris (four isolates), Dietzia natronolimnaea (two isolates), and Dietzia timorensis and Dietzia sp. (one isolate each). The eight Dietzia isolates were highly sensitive to 12 antimicrobial compounds. PMID:20220156

  8. PROmiRNA: a new miRNA promoter recognition method uncovers the complex regulation of intronic miRNAs

    PubMed Central

    2013-01-01

    The regulation of intragenic miRNAs by their own intronic promoters is one of the open problems of miRNA biogenesis. Here, we describe PROmiRNA, a new approach for miRNA promoter annotation based on a semi-supervised statistical model trained on deepCAGE data and sequence features. We validate our results with existing annotation, PolII occupancy data and read coverage from RNA-seq data. Compared to previous methods PROmiRNA increases the detection rate of intronic promoters by 30%, allowing us to perform a large-scale analysis of their genomic features, as well as elucidate their contribution to tissue-specific regulation. PROmiRNA can be downloaded from http://promirna.molgen.mpg.de. PMID:23958307

  9. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

  10. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

  11. miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy

    PubMed Central

    Myers, Jason R.; Gupta, Simone; Weng, Lien-Chun; Ashton, John M.; Cornish, Toby C.; Pandey, Akhilesh; Halushka, Marc K.

    2015-01-01

    Small RNA RNA-seq for microRNAs (miRNAs) is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM). Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA) are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench), miRge was faster (4 to 32-fold) and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html. PMID:26571139

  12. Biological activities of some Acacia spp. (Fabaceae) against new clinical isolates identified by ribosomal RNA gene-based phylogenetic analysis.

    PubMed

    Mahmoud, Mahmoud Fawzy; Alrumman, Sulaiman Abdullah; Hesham, Abd El-Latif

    2016-01-01

    Nowadays,most of the pathogenic bacteria become resistant to antibiotics. Therefore,the pharmaceutical properties of the natural plant extracts have become of interest to researchers as alternative antimicrobial agents. In this study,antibacterial activities of extract gained from Acacia etbaica, Acacia laeta, Acacia origena and Acacia pycnantha have been evaluated against isolated pathogenic bacteria (Strains MFM-01, MFM-10 and AH-09) using agar well diffusion methods.The bacterial strains were isolated from infected individuals,and their exact identification was detected on the basis of 16S rRNA gene amplification and sequence determination. Alignment results and the comparison of 16 SrRN A gene sequences of the isolates to 16 SrRN A gene sequences available in Gen Bank data base as well as the phylogenetic analysis confirmed the accurate position of the isolates as Klebsiella oxytoca strain MFM-01, Staphylococcus aureus strain MFM-10 and Klebsiella pneumoniae strain AH-09. Except for cold water, all tested solvents (Chloroform, petroleum ether, methanol, diethyl ether, and acetone) showed variation in their activity against studied bacteria. GC-MS analysis of ethanol extracts showed that four investigated Acacia species have different phyto components. Eight important pharmaceutical components were found in the legume of Acacia etbaica, seven in the legume of Acacia laeta, fifteen in the legume of Acacia origena and nine in the leaves of Acacia pycnantha. A dendrogram was constructed based on chemical composition, revealed that Acacia laeta is more closely related to Acacia etbaica forming on eclade, whereas Acacia origena less similar to other species. Our results demonstrated that, investigated plants and chemical compounds present could be used as promising antibacterial agents. PMID:26826814

  13. Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

    2011-01-01

    Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

  14. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    PubMed Central

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-01-01

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV) 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. PMID:26389898

  15. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1.

    PubMed

    Dong, Yangchao; Yang, Jing; Ye, Wei; Wang, Yuan; Ye, Chuantao; Weng, Daihui; Gao, Huan; Zhang, Fanglin; Xu, Zhikai; Lei, Yingfeng

    2015-01-01

    Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs). The streptavidin-binding aptamer S1 sequence was inserted into the 3' end of dengue virus (DENV) 5'-3' UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP) assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions. PMID:26389898

  16. Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

    PubMed Central

    Himmelfarb, H J; Simpson, E M; Friesen, J D

    1987-01-01

    Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain. PMID:3299061

  17. RNA-Based Stable Isotope Probing and Isolation of Anaerobic Benzene-Degrading Bacteria from Gasoline-Contaminated Groundwater

    PubMed Central

    Kasai, Yuki; Takahata, Yoh; Manefield, Mike; Watanabe, Kazuya

    2006-01-01

    Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [13C6]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the 13C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [14C]benzene showed that the labeled carbon was mostly incorporated into 14CO2 within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function. PMID:16672506

  18. Simultaneous isolation of high-quality DNA, RNA, miRNA and proteins from tissues for genomic applications

    PubMed Central

    Peña-Llopis, Samuel; Brugarolas, James

    2014-01-01

    Genomic technologies have revolutionized our understanding of complex Mendelian diseases and cancer. Solid tumors present several challenges for genomic analyses, such as tumor heterogeneity and tumor contamination with surrounding stroma and infiltrating lymphocytes. We developed a protocol to (i) select tissues of high cellular purity on the basis of histological analyses of immediately flanking sections and (ii) simultaneously extract genomic DNA (gDNA), messenger RNA (mRNA), noncoding RNA (ncRNA; enriched in microRNA (miRNA)) and protein from the same tissues. After tissue selection, about 12–16 extractions of DNA/RNA/protein can be obtained per day. Compared with other similar approaches, this fast and reliable methodology allowed us to identify mutations in tumors with remarkable sensitivity and to perform integrative analyses of whole-genome and exome data sets, DNA copy numbers (by single-nucleotide polymorphism (SNP) arrays), gene expression data (by transcriptome profiling and quantitative PCR (qPCR)) and protein levels (by western blotting and immunohistochemical analysis) from the same samples. Although we focused on renal cell carcinoma, this protocol may be adapted with minor changes to any human or animal tissue to obtain high-quality and high-yield nucleic acids and proteins. PMID:24136348

  19. Isolation, structure and expression of mammalian genes for histidyl-tRNA synthetase.

    PubMed Central

    Tsui, F W; Siminovitch, L

    1987-01-01

    A full length cDNA clone that codes for human histidyl-tRNA synthetase (HRS) and cDNA clones that span the full length transcript of hamster HRS have been isolated. The full length human HRS cDNA was expressed after transfection into Cos 1 cells and a CHO ts mutant defective in the gene for HRS. The complete nucleotide sequence of the hamster and human gene were obtained and extensive homologies were observed in three regions on comparing these sequences between themselves and with the sequence of HRS derived from yeast. These results provide unequivocal evidence that we have indeed cloned the hamster and human gene for HRS. Three overlapping phage recombinants containing the complete hamster chromosomal gene for HRS have also been isolated. The genomic HRS is divided into 13 exons. The precise locations of each of the 5' and 3' exon-intron boundaries were defined by sequencing the appropriate regions of the cloned genomic DNA and aligning them with the sequence of HRS cDNAs. These studies provide the basis for future structural and functional analysis of the gene for HRS. In particular, it will be of interest to examine if different exons of HRS correlate to different domains of the HRS polypeptide. Images PMID:3554142

  20. Avian pneumovirus (APV) RNA from wild and sentinel birds in the United States has genetic homology with RNA from APV isolates from domestic turkeys.

    PubMed

    Shin, H J; Njenga, M K; McComb, B; Halvorson, D A; Nagaraja, K V

    2000-11-01

    Nasal turbinates or swabs were collected from wild ducks, geese, owls, sparrows, swallows, and starlings and from sentinel ducks placed next to turkey farms experiencing avian pneumovirus (APV) infections and were analyzed for APV genome and infectious particles. APV RNA was detected in samples examined from geese, sparrows, and starlings. APV RNA and antibodies were also detected in two different groups of sentinel ducks. Infectious APV was recovered from sentinel duck samples. The APV M gene isolated from the wild birds had over 96% predicted amino acid identity with APV/Minnesota 2A, which was isolated earlier from domestic turkeys showing respiratory illness, suggesting that wild birds may be involved in spreading APV infection. PMID:11060113

  1. Isolation and analysis of (Gp)nXp sequences of rat liver 5S RNA by means of restricted ribonuclease T2 hydrolysis

    PubMed Central

    Willems, R.; Avdonina, T.; Lund, A.; Kisselev, L.L.

    1974-01-01

    Essentual difficulties arise when base number in oligoguanylic blocks and location of these blocks along the polynucleotide chain need to be determined in the course of determination of the nucleotide sequences in ribonucleic acids. To overcome this difficulty it is suggested to take advantage of a recently discovered resistance of phosphodiester bond between kethoxalated G and its 3′-neighbour against T2 RNase hydrolysis 1,2. The approach is illustrated by analysis of 5S RNA from rat liver. Sequences of general formula (Gp)nXp were isolated from T2 RNase hydrolysate of 5 S RNA rapidly and quantitatively. The information obtained greatly facilitates the whole procedure of sequencing. It is expected that the method proposed would be effective for analysis of 5 S and 4 S RNA and for highmolecular weight fragments of ribosomal and viral RNAs. PMID:4453523

  2. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    PubMed

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods. PMID:25629590

  3. Improved methods for typing nontypeable isolates of group B streptococci.

    PubMed

    Benson, Jeffrey A; Flores, Aurea E; Baker, Carol J; Hillier, Sharon L; Ferrieri, Patricia

    2002-06-01

    Group B streptococci (GBS) are classified by capsular polysaccharide (CPS) type and by cell surface-expressed proteins (c and R). Isolates lacking detectable CPS are considered nontypeable (NT) although they frequently express surface proteins. Immunological and genetic methods were used to study 91 NT GBS isolates collected during surveillance studies for invasive disease or colonization in pregnant or non-pregnant women and neonates less than seven days of age. CPS production was upregulated by the addition of glucose and sodium phosphate to Todd-Hewitt broth (THB) and cells were extracted using hot HCl or mutanolysin. Extracts were tested with antisera for specific CPS types Ia, Ib, and II - VIII by double immunodiffusion (DD) in agarose. By mutanolysin extraction, 12 (13.2%) of the 91 isolates were typeable. In contrast, only four of these 12 newly typeable isolates tested positive for CPS with the HCl extracts of cells grown in modified THB. DNA was analyzed by pulsed-field gel electrophoresis (PFGE) using SmaI restriction with NT isolates grouped by protein profile to facilitate analysis. PFGE results of the NT isolates were compared to DNA profiles of typeable isolates and were correlated with the DD results. The DNA profiles of the newly typeable isolates were similar to profiles of isolates with corresponding defined CPS type. Of the remaining 78 NT isolates digested by SmaI, 63 (80.8%) had DNA profiles that resembled those of specific types of GBS. These approaches will be useful for classification of NT isolates in continued epidemiological surveillance associated with GBS vaccine trials. PMID:12139427

  4. RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates

    PubMed Central

    Hazen, Tracy H.; Daugherty, Sean C.; Shetty, Amol; Mahurkar, Anup A.; White, Owen; Kaper, James B.; Rasko, David A.

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis. PMID:26124752

  5. Mitochondrial Structure and Function Are Disrupted by Standard Isolation Methods

    PubMed Central

    Picard, Martin; Taivassalo, Tanja; Ritchie, Darmyn; Wright, Kathryn J.; Thomas, Melissa M.; Romestaing, Caroline; Hepple, Russell T.

    2011-01-01

    Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca2+ handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca2+ challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H2O2 production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented. PMID:21512578

  6. Amplification-based method for microRNA detection.

    PubMed

    Shen, Yanting; Tian, Fei; Chen, Zhenzhu; Li, Rui; Ge, Qinyu; Lu, Zuhong

    2015-09-15

    Over the last two decades, the study of miRNAs has attracted tremendous attention since they regulate gene expression post-transcriptionally and have been demonstrated to be dysregulated in many diseases. Detection methods with higher sensitivity, specificity and selectivity between precursors and mature microRNAs are urgently needed and widely studied. This review gave an overview of the amplification-based technologies including traditional methods, current modified methods and the cross-platforms of them combined with other techniques. Many progresses were found in the modified amplification-based microRNA detection methods, while traditional platforms could not be replaced until now. Several sample-specific normalizers had been validated, suggesting that the different normalizers should be established for different sample types and the combination of several normalizers might be more appropriate than a single universal normalizer. This systematic overview would be useful to provide comprehensive information for subsequent related studies and could reduce the un-necessary repetition in the future. PMID:25930002

  7. Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

    PubMed

    Grayson, T H; Alexander, S M; Cooper, L F; Gilpin, M L

    2000-07-01

    The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries. PMID:11016696

  8. Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues.

    PubMed

    Filippova, J A; Stepanov, G A; Semenov, D V; Koval, O A; Kuligina, E V; Rabinov, I V; Richter, V A

    2015-01-01

    Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2'-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2'-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2'-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2'-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2'-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2'-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans. PMID:26085946

  9. SimRNA: a coarse-grained method for RNA folding simulations and 3D structure prediction.

    PubMed

    Boniecki, Michal J; Lach, Grzegorz; Dawson, Wayne K; Tomala, Konrad; Lukasz, Pawel; Soltysinski, Tomasz; Rother, Kristian M; Bujnicki, Janusz M

    2016-04-20

    RNA molecules play fundamental roles in cellular processes. Their function and interactions with other biomolecules are dependent on the ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. Here, we present SimRNA: a new method for computational RNA 3D structure prediction, which uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information, and, on established test sequences, it recapitulates secondary structure with high accuracy, including correct prediction of pseudoknots. For modeling of complex 3D structures, it can use additional restraints, derived from experimental or computational analyses, including information about secondary structure and/or long-range contacts. SimRNA also can be used to analyze conformational landscapes and identify potential alternative structures. PMID:26687716

  10. SimRNA: a coarse-grained method for RNA folding simulations and 3D structure prediction

    PubMed Central

    Boniecki, Michal J.; Lach, Grzegorz; Dawson, Wayne K.; Tomala, Konrad; Lukasz, Pawel; Soltysinski, Tomasz; Rother, Kristian M.; Bujnicki, Janusz M.

    2016-01-01

    RNA molecules play fundamental roles in cellular processes. Their function and interactions with other biomolecules are dependent on the ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. Here, we present SimRNA: a new method for computational RNA 3D structure prediction, which uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information, and, on established test sequences, it recapitulates secondary structure with high accuracy, including correct prediction of pseudoknots. For modeling of complex 3D structures, it can use additional restraints, derived from experimental or computational analyses, including information about secondary structure and/or long-range contacts. SimRNA also can be used to analyze conformational landscapes and identify potential alternative structures. PMID:26687716

  11. Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis.

    PubMed Central

    Chatellier, S; Huet, H; Kenzi, S; Rosenau, A; Geslin, P; Quentin, R

    1996-01-01

    The genetic diversity of a collection of 54 unrelated Streptococcus agalactiae strains isolated from the cerebrospinal fluid of neonates and of 60 unrelated carrier strains was evaluated by investigating the restriction fragment length polymorphism of the rRNA gene region. Three restriction enzymes were selected for use: PstI, HindIII, and CfoI. Clustering analysis revealed two phylogenetic groups of strains with 40% divergence. Group I contained two clusters, A and B, and group II contained three clusters, C, D, and E. Strains of serotype Ia were mostly distributed in cluster A, and strains of serotype Ib were mostly distributed in cluster E. Serotype III isolates did not cluster. Nevertheless, 37 of 39 isolates belonging to cluster B were serotype III. With HindIII, two rRNA gene banding patterns characterized 38 of the 39 strains of cluster B, which represents a high-virulence group. In addition, two rRNA gene banding patterns with each enzyme and/or a pair of CfoI fragments of 905 and 990 bp identified 81% of the invasive strains. On account of the genetic homogeneity of the cerebrospinal fluid strains, ribotyping is a powerful typing method for investigation of nosocomial or epidemic invasive infections only when all three enzymes are used or when PstI and HindIII or PstI and CfoI are combined with serotyping (index of discrimination, > 0.95). PMID:8897176

  12. Updated 16S rRNA-RFLP method for the identification of all currently characterised Arcobacter spp

    PubMed Central

    2012-01-01

    Background Arcobacter spp. (family Campylobacteraceae) are ubiquitous zoonotic bacteria that are being increasingly recognised as a threat to human health. A previously published 16S rRNA-RFLP Arcobacter spp. identification method produced specific RFLP patterns for the six species described at that time, using a single endonuclease (MseI). The number of characterised Arcobacter species has since risen to 17. The aim of the present study was to update the 16S rRNA-RFLP identification method to include all currently characterised species of Arcobacter. Results Digestion of the 16S rRNA gene with the endonuclease MseI produced clear, distinctive patterns for 10 of the 17 species, while the remaining species shared a common or very similar RFLP pattern. Subsequent digestion of the 16S rRNA gene from these species with the endonucleases MnlI and/or BfaI generated species-specific RFLP patterns. Conclusions 16S rRNA-RFLP analysis identified 17 Arcobacter spp. using either polyacrylamide or agarose gel electrophoresis. Microheterogeneities within the 16S rRNA gene, which interfered with the RFLP identification, were also documented for the first time in this genus, particularly in strains of Arcobacter cryaerophilus isolated from animal faeces and aborted foetuses. PMID:23244705

  13. RNA imaging in living cells – methods and applications

    PubMed Central

    Urbanek, Martyna O; Galka-Marciniak, Paulina; Olejniczak, Marta; Krzyzosiak, Wlodzimierz J

    2014-01-01

    Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications. PMID:25483044

  14. The effect of cell subset isolation method on gene expression in leukocytes

    PubMed Central

    White, Cory; Lada, Steven; Du, Pinyi; Vaida, Florin; Blanco, Julià; Spina, Celsa A.; Woelk, Christopher H.

    2014-01-01

    Multiple scientific disciplines require the isolation of specific subsets of blood cells from patient samples for gene expression analysis by microarray or RNA-sequencing, preserving disease- or treatment-related signatures. However, little is known with respect to the impact of different cell isolation methods on gene expression and the effects of positive selection, negative selection and fluorescence activated cell sorting (FACS) have not previously been assessed in parallel. To address this knowledge gap, CD4+ T cells, CD8+ T cells, B cells and monocytes were isolated from blood samples from 5 independent donors using positive immunomagnetic selection, negative immunomagnetic selection and FACS. We hypothesized that positive selection and FACS would yield higher purity but may have an impact on gene expression since both methods utilize antibodies that bind surface receptors of the cell type of interest. Moreover, FACS might upregulate stress response genes due to passage of the cells through the sorter. Microarray gene expression data was generated and subjected to unsupervised clustering and differential gene expression analysis. Surprisingly, these analyses revealed that gene expression signatures were more similar between cells isolated by negative selection and FACS compared to cells isolated by positive selection. Moreover, genes that are involved in the response to stress generally had the highest expression in cells isolated by negative or positive selection and not FACS. Thus, FACS is the recommended method for isolation of leukocyte subsets for gene expression studies since this method results in the purest subset populations and does not appear to induce a stress response. PMID:24115734

  15. Isolation of an mRNA binding protein homologue that is expressed in nociceptors.

    PubMed

    Eilers, Helge; Trilk, Sharon L; Lee, Sook Young; Xue, Qing; Jong, Beverly E; Moff, Irene; Levine, Jon D; Schumacher, Mark A

    2004-11-01

    The peripheral detection of painful stimuli requires the activation of small-diameter primary afferent neurons known as nociceptors. We have exploited two features of nociceptor biology, expression of the high affinity receptor for nerve growth factor (TrkA) and sensitivity to capsaicin, to isolate novel proteins using a differential display cloning scheme. A resulting approximately 4.3-kb cDNA was isolated and sequence analysis inferred a approximately 157-kDa protein containing a signal/mitochondrial targeting peptide sequence. Due to its molecular weight and significant amino acid identity with 'human leucine-rich protein 130'[leucine-rich pentatricopeptide motif containing (LRPPRC)], we termed the cDNA candidate leucine-rich protein 157 (rLRP157). Western blot analysis of HEK293 cells over-expressing the candidate cDNA showed a single protein product of similar size to that found in rat dorsal root ganglion as well as in other neuronal tissues and cell lines. Although expressed in a wide variety of tissues, in situ hybridization and immunohistochemistry in dorsal root ganglion revealed that rLRP157 expression was restricted to the small-diameter neurons. Sequence identity with previously characterized mRNA binding proteins and its subcellular localization in sensory neurons suggest that rLRP157 is associated with mitochondrial function. Moreover, the genetic basis of French-Canadian Leigh syndrome, which confers a loss of mitochondrial cytochrome c oxidase and is characterized by neurodegeneration, was recently mapped to a mutation in the LRPPRC gene. Taken together with its expression in small-diameter sensory neurons, we hypothesize that rLRP157, the rat orthologue of the human LRPPRC, may play a role in the modulation of peripheral pain transduction and serve as a novel marker for nociceptor subtypes. PMID:15525270

  16. Rapid method for the isolation of mammalian sperm DNA

    PubMed Central

    Wu, Haotian; de Gannes, Matthew K.; Luchetti, Gianna; Pilsner, J. Richard

    2015-01-01

    The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist. PMID:26054765

  17. Rapid method for the isolation of mammalian sperm DNA.

    PubMed

    Wu, Haotian; de Gannes, Matthew K; Luchetti, Gianna; Pilsner, J Richard

    2015-06-01

    The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist. PMID:26054765

  18. An automatic and effective tooth isolation method for dental radiographs

    NASA Astrophysics Data System (ADS)

    Lin, P.-L.; Huang, P.-W.; Cho, Y. S.; Kuo, C.-H.

    2013-03-01

    Tooth isolation is a very important step for both computer-aided dental diagnosis and automatic dental identification systems, because it will directly affect the accuracy of feature extraction and, thereby, the final results of both types of systems. This paper presents an effective and fully automatic tooth isolation method for dental X-ray images, which contains up-per-lower jaw separation, single tooth isolation, over-segmentation verification, and under-segmentation detection. The upper-lower jaw separation mechanism is based on a gray-scale integral projection to avoid possible information loss and incorporates with the angle adjustment to handle skewed images. In a single tooth isolation, an adaptive windowing scheme for locating gap valleys is proposed to improve the accuracy. In over-segmentation, an isolation-curve verification scheme is proposed to remove excessive curves; and in under-segmentation, a missing-teeth detection scheme is proposed. The experimental results demonstrate that our method achieves the accuracy rates of 95.63% and 98.71% for the upper and lower jaw images, respectively, from the test database of 60 bitewing dental radiographs, and performs better for images with severe teeth occlusion, excessive dental works, and uneven illumination than that of Nomir and Abdel-Mottaleb's method. The method without upper-lower jaw separation step also works well for panoramic and periapical images.

  19. A convenient method for saponin isolation in tumour therapy.

    PubMed

    Weng, Alexander; Jenett-Siems, Kristina; Schmieder, Peter; Bachran, Diana; Bachran, Christopher; Görick, Cornelia; Thakur, Mayank; Fuchs, Hendrik; Melzig, Matthias F

    2010-03-01

    Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy. PMID:20144565

  20. Well fluid isolation and sample apparatus and method

    DOEpatents

    Schalla, Ronald; Smith, Ronald M.; Hall, Stephen H.; Smart, John E.

    1995-01-01

    The present invention specifically permits purging and/or sampling of a well but only removing, at most, about 25% of the fluid volume compared to conventional methods and, at a minimum, removing none of the fluid volume from the well. The invention is an isolation assembly that is inserted into the well. The isolation assembly is designed so that only a volume of fluid between the outside diameter of the isolation assembly and the inside diameter of the well over a fluid column height from the bottom of the well to the top of the active portion (lower annulus) is removed. A seal may be positioned above the active portion thereby sealing the well and preventing any mixing or contamination of inlet fluid with fluid above the packer. Purged well fluid is stored in a riser above the packer. Ports in the wall of the isolation assembly permit purging and sampling of the lower annulus along the height of the active portion.

  1. Elevation in heat shock protein 72 mRNA following contractions in isolated single skeletal muscle fibers.

    PubMed

    Stary, Creed M; Walsh, Brandon J; Knapp, Amy E; Brafman, David; Hogan, Michael C

    2008-08-01

    The purpose of the present study was 1) to develop a stable model for measuring contraction-induced elevations in mRNA in single skeletal muscle fibers and 2) to utilize this model to investigate the response of heat shock protein 72 (HSP72) mRNA following an acute bout of fatiguing contractions. Living, intact skeletal muscle fibers were microdissected from lumbrical muscle of Xenopus laevis and either electrically stimulated for 15 min of tetanic contractions (EX; n=26) or not stimulated to contract (REST; n=14). The relative mean developed tension of EX fibers decreased to 29+/-7% of initial peak tension at the stimulation end point. Following treatment, individual fibers were allowed to recover for 1 (n=9), 2 (n=8), or 4 h (n=9) prior to isolation of total cellular mRNA. HSP72, HSP60, and cardiac alpha-actin mRNA content were then assessed in individual fibers using quantitative PCR detection. Relative HSP72 mRNA content was significantly (P<0.05) elevated at the 2-h postcontraction time point relative to REST fibers when normalized to either HSP60 (18.5+/-7.5-fold) or cardiac alpha-actin (14.7+/-4.3-fold), although not at the 1- or 4-h time points. These data indicate that 1) extraction of RNA followed by relative quantification of mRNA of select genes in isolated single skeletal muscle fibers can be reliably performed, 2) HSP60 and cardiac alpha-actin are suitable endogenous normalizing genes in skeletal muscle following contractions, and 3) a significantly elevated content of HSP72 mRNA is detectable in skeletal muscle 2 h after a single bout of fatiguing contractions, despite minimal temperature changes and without influence from extracellular sources. PMID:18525012

  2. Detection of RNA-DNA association by a proximity ligation-based method.

    PubMed

    Petruk, Svetlana; Fenstermaker, Tyler K; Black, Kathryn L; Brock, Hugh W; Mazo, Alexander

    2016-01-01

    We describe a proximity ligation assay (PLA)-based method of assessing association of DNA and RNA in single cells during the cell cycle. Pulse-labeling of DNA with EdU and RNA with BrU and testing their close proximity by PLA demonstrates that RNA synthesis in individual cells resumes about 30-45 min after DNA replication. Consistent with this conclusion, RNA Pol II phosphorylated at Ser2 of its CTD is detected at the same time as RNA transcripts on nascent DNA. Our results also show that RNA is associated with DNA foci during all stages of mitosis. PMID:27256324

  3. Detection of RNA-DNA association by a proximity ligation-based method

    PubMed Central

    Petruk, Svetlana; Fenstermaker, Tyler K.; Black, Kathryn L.; Brock, Hugh W.; Mazo, Alexander

    2016-01-01

    We describe a proximity ligation assay (PLA)-based method of assessing association of DNA and RNA in single cells during the cell cycle. Pulse-labeling of DNA with EdU and RNA with BrU and testing their close proximity by PLA demonstrates that RNA synthesis in individual cells resumes about 30–45 min after DNA replication. Consistent with this conclusion, RNA Pol II phosphorylated at Ser2 of its CTD is detected at the same time as RNA transcripts on nascent DNA. Our results also show that RNA is associated with DNA foci during all stages of mitosis. PMID:27256324

  4. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

    PubMed

    Taylor, Douglas D; Shah, Sahil

    2015-10-01

    Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and

  5. Isolate Speech Recognition Based on Time-Frequency Analysis Methods

    NASA Astrophysics Data System (ADS)

    Mantilla-Caeiros, Alfredo; Nakano Miyatake, Mariko; Perez-Meana, Hector

    A feature extraction method for isolate speech recognition is proposed, which is based on a time frequency analysis using a critical band concept similar to that performed in the inner ear model; which emulates the inner ear behavior by performing signal decomposition, similar to carried out by the basilar membrane. Evaluation results show that the proposed method performs better than other previously proposed feature extraction methods when it is used to characterize normal as well as esophageal speech signal.

  6. Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    SciTech Connect

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason W.; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2015-10-20

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  7. Methods and composition for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason W.; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2012-05-08

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  8. Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

    DOEpatents

    Schultz, Peter; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2006-08-01

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  9. Isolation and identification of lactic acid bacteria from Tarag in Eastern Inner Mongolia of China by 16S rRNA sequences and DGGE analysis.

    PubMed

    Liu, Wenjun; Bao, Qiuhua; Jirimutu; Qing, Manjun; Siriguleng; Chen, Xia; Sun, Ting; Li, Meihua; Zhang, Jiachao; Yu, Jie; Bilige, Menghe; Sun, Tiansong; Zhang, Heping

    2012-01-20

    Tarag is a characteristic fermented dairy product with rich microflora (especially lactic acid bacteria), developed by the people of Mongolian nationality in Inner Mongolia of China and Mongolia throughout history. One hundred and ninety-eight samples of Tarag were collected from scattered households in Eastern Inner Mongolia, and total of 790 isolates of lactic acid bacteria (LAB) were isolated by traditional pure culture method. To identify these isolates and analyze their biodiversity, 16S rRNA gene sequences analysis and PCR-DGGE were performed respectively. The results showed that 790 isolates could be classified as 31 species and subspecies. Among these isolates, Lactobacillus helveticus (153 strains, about 19.4%), Lactococcus lactis subsp. lactis (132 strains, about 16.7%) and Lactobacillus casei (106 strains, about 11.0%) were considered as the predominated species in the traditional fermented dairy products (Tarag) in Eastern Inner Mongolia. It was shown that the biodiversity of LAB in Tarag in Inner Mongolia was very abundant, and this traditional fermented dairy product could be considered as valuable resources for LAB isolation and probiotic selection. PMID:21689912

  10. Isolation of a novel RNA-dependent RNA polymerase 6 from Nicotiana glutinosa, NgRDR6, and analysis of its response to biotic and abiotic stresses.

    PubMed

    Yang, Haifang; Wang, Mian; Gao, Zheng; Zhu, Changxiang; Guo, Xingqi

    2011-02-01

    RNA-dependent RNA polymerases (RDRs) play an important role in RNA silencing, antiviral and developmental progress. Here, we firstly isolated the full-length cDNA, genomic DNA and 5'-flanking region of RDR6 from Nicotiana glutinosa (NgRDR6). Sequences analysis revealed that the cDNA of NgRDR6 was 3,921 bp in length, and the deduced protein consisted of 1,197 amino acids, containing all highly conserved sequence motifs that are present among all RDRs families. Moreover, two introns were detected in the genomic sequences. We also firstly investigated the expression profiles of plant RDR6 under the treatments of gibberellin A (GA), H(2)O(2,) methyl jasmonate (MeJA), Potato virus Y (PVY), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Rhizoctonia Solani and Colletotrichum nicotianae. In addition, the expression patterns of RDR6 in Nicotiana glutinosa under the treatments of salicylic acid (SA) and abscisic acid (ABA) were also been analyzed. The results indicated that the NgRDR6 mRNA accumulation could be induced by ABA, GA, MeJA, CMV, Rhizoctonia Solani and Colletotrichum nicotianae. In contrast, the expression level of NgRDR6 exhibited no remarkable difference under the treatments of PVY, TMV, H(2)O(2) and SA. Further investigation suggested several potential cis-acting elements were found in the 5'-flanking sequence of NgRDR6, which might be responsible for the enhanced response to phytohormones. PMID:20495874

  11. RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA.

    PubMed

    Ferriol, I; Turina, M; Zamora-Macorra, E J; Falk, B W

    2016-05-01

    Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA. PMID:26756828

  12. A simple method for construction of artificial microRNA vector in plant.

    PubMed

    Li, Yang; Li, Yang; Zhao, Sunping; Zhong, Sheng; Wang, Zhaohai; Ding, Bo; Li, Yangsheng

    2014-10-01

    Artificial microRNA (amiRNA) is a powerful tool for silencing genes in many plant species. Here we provide an easy method to construct amiRNA vectors that reinvents the Golden Gate cloning approach and features a novel system called top speed amiRNA construction (TAC). This speedy approach accomplishes one restriction-ligation step in only 5 min, allowing easy and high-throughput vector construction. Three primers were annealed to be a specific adaptor, then digested and ligated on our novel vector pTAC. Importantly, this method allows the recombined amiRNA constructs to maintain the precursor of osa-miR528 with exception of the desired amiRNA/amiRNA* sequences. Using this method, our results showed the expected decrease of targeted genes in Nicotiana benthamiana and Oryza sativa. PMID:24930106

  13. An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

    PubMed Central

    Holmes, Ashleigh; Birse, Louise; Jackson, Robert W.; Holden, Nicola J.

    2014-01-01

    Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety. PMID:25018749

  14. A rapid and sensitive nonradioactive method applicable for genome-wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

    PubMed Central

    Wu, Jingyan; Huang, Hsiao-Yun; Hopper, Anita K.

    2013-01-01

    The conventional small RNA isolation and detection methods for yeast cells have been designed for a small number of samples. In order to conduct a genome-wide assessment of how each gene product impacts upon small non-coding RNAs, we developed a rapid method for analyzing small RNAs from Saccharomyces cerevisiae wild-type and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive nonradioactive Northern method for RNA detection. The RNA isolation procedure is highly reproducible and requires only 4 hours for processing 96 samples, and yields RNA of good quality and quantity. The nonradioactive Northern method employs digoxigenin (DIG)-labeled DNA probes and chemiluminescence. It detects femtomole-level small RNAs within 1-minute exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and re-probing procedures for analysis of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe, and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking, and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. PMID:23417998

  15. Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

    PubMed

    Singh, Deepali; Boeras, Ioana; Singh, Gatikrushna; Boris-Lawrie, Kathleen

    2016-01-01

    All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification. PMID:26714709

  16. High rates of double-stranded RNA viruses and Mycoplasma hominis in Trichomonas vaginalis clinical isolates in South Brazil.

    PubMed

    da Luz Becker, Débora; dos Santos, Odelta; Frasson, Amanda Piccoli; de Vargas Rigo, Graziela; Macedo, Alexandre José; Tasca, Tiana

    2015-08-01

    Trichomonas vaginalis is the etiological agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in world, with 276.4 million new cases each year. T. vaginalis can be naturally infected with Mycoplasma hominis and Trichomonasvirus species. This study aimed to evaluate the prevalence of T. vaginalis infected with four distinct T. vaginalis viruses (TVVs) and M. hominis among isolates from patients in Porto Alegre city, South Brazil. An additional goal of this study was to investigate whether there is association between metronidazole resistance and the presence of M. hominis during TVV infection. The RNA expression level of the pyruvate ferredoxin oxidoreductase (PFOR) gene was also evaluated among metronidazole-resistant and metronidazole-sensitive T. vaginalis isolates. A total of 530 urine samples were evaluated, and 5.7% samples were positive for T. vaginalis infection. Among them, 4.51% were isolated from female patients and 1.12% were from male patients. Remarkably, the prevalence rates of M. hominis and TVV-positive T. vaginalis isolates were 56.7% and 90%, respectively. Most of the T. vaginalis isolates were metronidazole-sensitive (86.7%), and only four isolates (13.3%) were resistant. There is no statistically significant association between infection by M. hominis and infection by TVVs. Our results refute the hypothesis that the presence of the M. hominis and TVVs is enough to confer metronidazole resistance to T. vaginalis isolates. Additionally, the role of PFOR RNA expression levels in metronidazole resistance as the main mechanism of resistance to metronidazole could not be established. This study is the first report of the T. vaginalis infection by M. hominis and TVVs in a large collection of isolates from South Brazil. PMID:26160539

  17. Comparison of two methods for RNA extraction from the nucleus pulposus of intervertebral discs.

    PubMed

    Gan, M F; Yang, H L; Qian, J L; Wu, C S; Yuan, C X; Li, X F; Zou, J

    2016-01-01

    RNA extraction from the nucleus pulposus of intervertebral discs has been extensively used in orthopedic studies. We compared two methods for extracting RNA from the nucleus pulposus: liquid nitrogen grinding and enzyme digestion. The RNA was detected by agarose gel electrophoresis, and the purity was evaluated by absorbance ratio using a spectrophotometer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Thirty human lumbar intervertebral discs were used in this study. The liquid nitrogen-grinding method was used for RNA extraction from 15 samples, and the mean RNA concentration was 491.04 ± 44.16 ng/mL. The enzyme digestion method was used on 15 samples, and the mean RNA concentration was 898.42 ± 38.64 ng/mL. The statistical analysis revealed that there was a significant difference in concentration between the different methods. Apparent 28S, 18S, and 5S bands were detectable in RNA extracted using the enzyme digestion method, whereas no 28S or 18S bands were detected in RNA extracted using the liquid nitrogen-grinding method. The GAPDH band was visible, and no non-specific band was detected in the RT-PCR assay by the enzyme digestion method. Therefore, the enzyme digestion method is an efficient and easy method for RNA extraction from the nucleus pulposus of intervertebral discs for further intervertebral disc degeneration-related studies. PMID:27323116

  18. New Rapid Method of DNA Isolation from Milk Somatic Cells

    PubMed Central

    Pokorska, Joanna; Kułaj, Dominika; Dusza, Magdalena; Żychlińska-Buczek, Justyna; Makulska, Joanna

    2016-01-01

    ABSTRACT Isolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses. PMID:26913552

  19. New Rapid Method of DNA Isolation from Milk Somatic Cells.

    PubMed

    Pokorska, Joanna; Kułaj, Dominika; Dusza, Magdalena; Żychlińska-Buczek, Justyna; Makulska, Joanna

    2016-04-01

    Isolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses. PMID:26913552

  20. A novel dsRNA element isolated from the Aspergillus foetidus mycovirus complex.

    PubMed

    Kozlakidis, Zisis; Herrero, Noemi; Ozkan, Selin; Bhatti, Muhammad F; Coutts, Robert H A

    2013-12-01

    Aspergillus foetidus virus (AfV) contains at least two icosahedral particle types named AfV-fast (-F) and AfV-slow (-S), based on relative electrophoretic mobility. AfV-F is a quadripartite double-stranded RNA (dsRNA) virus, and AfV-S contains AfV-S1, which is a member of the genus Victorivirus in the family Totiviridae, and AfV-S2, which may be a satellite RNA or satellite virus and is described here. Analysis of the complete AfV-S2 nucleotide sequence reveals it to be significantly similar to an unclassified RNA from the fungus Rosellinia necatrix and distantly related to the RNA-dependent RNA polymerases of several single-stranded RNA genomes. PMID:23827976

  1. Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification.

    PubMed

    Chetverina, Helena V; Falaleeva, Marina V; Chetverin, Alexander B

    2004-11-15

    A universal procedure that permits the whole human blood to be tested for the presence of single molecules of DNA and RNA targets is described. The procedure includes a novel protocol for the isolation of total nucleic acids from the guanidinium thiocyanate lysate of unfractionated blood in which, prior to phenol/chloroform extraction, the sample is deproteinized by precipitation with isopropanol. The procedure results in a nearly 100% yield of DNA and RNA, preserves the integrity of RNA, and removes any polymerase chain reaction (PCR) inhibitors. Following reverse transcription (RT), target molecules are counted after having been amplified as molecular colonies by carrying out PCR in a polyacrylamide gel. The entire procedure was checked by assaying viral DNA and RNA in 100-microl aliquots of the whole blood and was found to be capable of detecting 100% molecules of DNA target and 50% molecules of RNA target. Unexpectedly, nucleic acids at relatively high concentrations (1 ng/microl) were found to selectively inhibit the RT activity of Thermus thermophilus DNA polymerase without affecting its DNA-dependent polymerization activity. It follows that the popular single-enzyme RT-PCR format, in which this DNA polymerase serves for both RT and PCR, is not appropriate for assaying rare RNA targets. PMID:15494145

  2. Methods to enable the design of bioactive small molecules targeting RNA

    PubMed Central

    Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

    2014-01-01

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  3. Methods to enable the design of bioactive small molecules targeting RNA.

    PubMed

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  4. An integrated miRNA functional screening and target validation method for organ morphogenesis

    PubMed Central

    Rebustini, Ivan T.; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A.; Maas, Richard L.

    2016-01-01

    The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs. PMID:26980315

  5. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences.

    PubMed

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-07-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences. PMID:26943621

  6. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences

    PubMed Central

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-01-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences. PMID:26943621

  7. Sequence Variation in the Small-Subunit rRNA Gene of Plasmodium malariae and Prevalence of Isolates with the Variant Sequence in Sichuan, China

    PubMed Central

    Liu, Qing; Zhu, Shenghua; Mizuno, Sahoko; Kimura, Masatsugu; Liu, Peina; Isomura, Shin; Wang, Xingzhen; Kawamoto, Fumihiko

    1998-01-01

    By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3′ region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia. PMID:9774600

  8. Direct PCR amplification of the 16S rRNA gene from single microbial cells isolated from an Antarctic iceberg using laser microdissection microscopy

    NASA Astrophysics Data System (ADS)

    Yanagihara, Katsuhiko; Niki, Hironori; Baba, Tomoya

    2011-09-01

    Here, we describe a technique that allows the genetic linage analysis of 16S rRNA genes in bacteria observed under a microscope. The technique includes the isolation of microbial cells using a laser microdissection microscope, lysis of the cells, and amplification of the 16S rRNA genes in the isolated cells without interference by bacterial DNA contamination from the experimental environment or reagents. Using this technique, we successfully determined 15 16S rRNA gene sequences in cells isolated from an Antarctic iceberg. These sequences showed 94%-100% identity to their closest strains, which included bacteria that occur in aqueous, marine, and soil environments.

  9. A fully enzymatic method for site-directed spin labeling of long RNA

    PubMed Central

    Lebars, Isabelle; Vileno, Bertrand; Bourbigot, Sarah; Turek, Philippe; Wolff, Philippe; Kieffer, Bruno

    2014-01-01

    Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies. PMID:24981512

  10. Lupin protein isolate versus casein modifies cholesterol excretion and mRNA expression of intestinal sterol transporters in a pig model

    PubMed Central

    2014-01-01

    Background Lupin proteins exert hypocholesterolemic effects in man and animals, although the underlying mechanism remains uncertain. Herein we investigated whether lupin proteins compared to casein modulate sterol excretion and mRNA expression of intestinal sterol transporters by use of pigs as an animal model with similar lipid metabolism as humans, and cellular cholesterol-uptake by Caco-2 cells. Methods Two groups of pigs were fed cholesterol-containing diets with either 230 g/kg of lupin protein isolate from L. angustifolius or 230 g/kg casein, for 4 weeks. Faeces were collected quantitatively over a 5 d period for analysis of neutral sterols and bile acids by gas chromatographically methods. The mRNA abundances of intestinal lipid transporters were analysed by real-time RT-PCR. Cholesterol-uptake studies were performed with Caco-2 cells that were incubated with lupin conglutin γ, phytate, ezetimibe or albumin in the presence of labelled [4-14C]-cholesterol. Results Pigs fed the lupin protein isolate revealed lower cholesterol concentrations in total plasma, LDL and HDL than pigs fed casein (P < 0.05). Analysis of faeces revealed a higher output of cholesterol in pigs that were fed lupin protein isolate compared to pigs that received casein (+57.1%; P < 0.05). Relative mRNA concentrations of intestinal sterol transporters involved in cholesterol absorption (Niemann-Pick C1-like 1, scavenger receptor class B, type 1) were lower in pigs fed lupin protein isolate than in those who received casein (P < 0.05). In vitro data showed that phytate was capable of reducing the uptake of labelled [4-14C]-cholesterol into the Caco-2 cells to the same extend as ezetimibe when compared to control (−20.5% vs. −21.1%; P < 0.05). Conclusions Data reveal that the cholesterol-lowering effect of lupin protein isolate is attributable to an increased faecal output of cholesterol and a reduced intestinal uptake of cholesterol. The findings indicate phytate as a

  11. Guard Cell Purification and RNA Isolation Suitable for High Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses

    PubMed Central

    Obulareddy, Nisita; Panchal, Shweta; Melotto, Maeli

    2014-01-01

    Stomata, the micro-pores on leaf surface, are formed by a pair of guard cells. In addition to control water loss and gas exchange between the plant and the environment, these cells act as immunity gates to prevent pathogen invasion of the plant apoplast. Here, we report a brief procedure to obtain highly pure guard cell preparations using conditions that preserve the guard cell transcriptome as much as possible for a robust high-throughput RNA sequence analysis. The advantages of this procedure included: 1) substantial shortening of the time required for obtaining high yield of >97% pure guard cell protoplasts (GCP), 2) extraction of enough amount of high quality RNA for direct sequencing, and 3) limited RNA decay during sample manipulation. Gene expression analysis by RT-qPCR revealed that wound-related genes were not induced during release of guard cells from leaves. To validate our approach, we performed a high throughput deep-sequencing of guard cell transcriptome (RNA-seq). A total of 18,994 nuclear-encoded transcripts was detected, which expanded the transcriptome by 70%. The optimized GCP isolation and RNA extraction protocols are simple, reproducible, and fast allowing the discovery of genes and regulatory networks inherent to the guard cells under various stresses. PMID:23634837

  12. RNA-Based Methods Increase the Detection of Fecal Bacteria and Fecal Identifiers in Environmental Waters

    EPA Science Inventory

    We evaluated the use of qPCR RNA-based methods in the detection of fecal bacteria in environmental waters. We showed that RNA methods can increase the detection of fecal bacteria in multiple water matrices. The data suggest that this is a viable alternative for the detection of a...

  13. Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection

    PubMed Central

    2014-01-01

    Background In order to provide gene expression profiles of different cell types, the primary step is to isolate the specific cells of interest via laser capture microdissection (LCM), followed by extraction of good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous gene expression studies on specific cell populations, providing valuable insights into specific cellular changes in diseases. However, such strategy imposed challenges as cells of interests are often available in limited quantities and quality of RNA may be compromised during long periods of time spent on collection of cells and extraction of total RNA; therefore, it is crucial that protocols for sample preparation should be optimised according to different cell populations. Findings We made several modifications to existing protocols to improve the total RNA yield and integrity for downstream qPCR analyses. A modified condensed hematoxylin and eosin (H&E) staining protocol was developed for the identification of rat renal proximal tubular cells (PTCs). It was then determined that a minimal of eight thousands renal PTCs were required to meet the minimal total RNA yield required for downstream qPCR. RNA integrity was assessed using at every progressive step of sample preparation. Therefore, we decided that the shortened H&E staining, together with microdissection should be performed consecutively within twenty minutes for good quality for gene expression analysis. These modified protocols were later applied on six individual rat samples. A panel of twenty rat renal drug transporters and five housekeeping genes showed Ct values below thirty-five, confirming the expression levels of these drug transporters can be detected. Conclusions We had successfully optimized the protocols to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR. This protocol may be

  14. Isolation and properties of an RNA fraction present in Brucella culture supernatants.

    PubMed Central

    Corbel, M. J.; Brewer, R. A.

    1980-01-01

    The supernatant fluids of batch and continuous cultures of Brucella strains contained up to 100 mg/l of soluble RNA which could be recovered by precipitation with lysozyme, This RNA fraction had many of the properties of ribosomal RNA and was single-stranded, sensitive to ribonuclease, with an approximate sedimentation constant of 5S, a molecular weight of about 35000 daltons and an adenine; guanine; cytosine; uracil content of 17.5; 26.5; 33; 23 mol% respectively. RNA fractions from lysozyme precipitates evoked high titres of Brucella agglutinins on injection into rabbits and induced acute inflammatory responses in guinea-pig skin. Highly purified RNA fractions prepared by phenol extraction of lysozyme precipitates did not evoke antibodies to Brucella abortus. Images Fig. 1 Fig. 2 Fig. 1 Fig. 2 Fig. 3 PMID:6153668

  15. Methods for identification of Staphylococcus aureus isolates in cases of bovine mastitis.

    PubMed

    Boerlin, Patrick; Kuhnert, Peter; Hüssy, Daniela; Schaellibaum, Melchior

    2003-02-01

    A total of 272 staphylococcal isolates from cases of bovine mastitis (159 Staphylococcus aureus) belonging to 12 different species were identified with ID32 STAPH galleries, and 51 of them were confirmed by 16S rRNA gene (rrs) sequencing. The same isolates were examined for their hemolytic activity on sheep blood agar, DNase activity, and coagulase activity and with two rapid identification kits (Slidex Staph Plus kit and RAPIDEC Staph from Bio-Merieux). The results of this study confirm those obtained by other groups with hemolysis, DNase, and coagulase. Only 50% of S. aureus isolates from mastitis cases show coagulase activity after 4 h of incubation, and a 24-h incubation is necessary for the full sensitivity of this test. In contrast to results from other studies with human isolates, the Slidex Staph Plus kit was not sensitive enough for the identification of S. aureus from bovine mastitis samples. The aurease test of the RAPIDEC Staph kit showed 100% sensitivity and 100% specificity. Used in conjunction with hemolysis patterns, the RAPIDEC Staph kit is therefore very well adapted to rapid, efficient, and cost-effective identification of S. aureus in cultures from bovine mastitis samples. Sequencing of rrs genes also proved very efficient in identifying the Staphylococcus species encountered in these samples and confirming phenotypical identification results with unsatisfactory scores. With continuously improving technologies and decreasing costs, genetic identification methods like rrs gene sequencing will soon find a place in routine veterinary diagnostics. PMID:12574280

  16. Methods for Identification of Staphylococcus aureus Isolates in Cases of Bovine Mastitis

    PubMed Central

    Boerlin, Patrick; Kuhnert, Peter; Hüssy, Daniela; Schaellibaum, Melchior

    2003-01-01

    A total of 272 staphylococcal isolates from cases of bovine mastitis (159 Staphylococcus aureus) belonging to 12 different species were identified with ID32 STAPH galleries, and 51 of them were confirmed by 16S rRNA gene (rrs) sequencing. The same isolates were examined for their hemolytic activity on sheep blood agar, DNase activity, and coagulase activity and with two rapid identification kits (Slidex Staph Plus kit and RAPIDEC Staph from Bio-Merieux). The results of this study confirm those obtained by other groups with hemolysis, DNase, and coagulase. Only 50% of S. aureus isolates from mastitis cases show coagulase activity after 4 h of incubation, and a 24-h incubation is necessary for the full sensitivity of this test. In contrast to results from other studies with human isolates, the Slidex Staph Plus kit was not sensitive enough for the identification of S. aureus from bovine mastitis samples. The aurease test of the RAPIDEC Staph kit showed 100% sensitivity and 100% specificity. Used in conjunction with hemolysis patterns, the RAPIDEC Staph kit is therefore very well adapted to rapid, efficient, and cost-effective identification of S. aureus in cultures from bovine mastitis samples. Sequencing of rrs genes also proved very efficient in identifying the Staphylococcus species encountered in these samples and confirming phenotypical identification results with unsatisfactory scores. With continuously improving technologies and decreasing costs, genetic identification methods like rrs gene sequencing will soon find a place in routine veterinary diagnostics. PMID:12574280

  17. Effect of RNA Integrity Determined With the Agilent 2100 Bioanalyzer on Bacterial RNA Quantification with RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA integrity is critical for successful RNA quantification. The level of integrity required differs among sources and extraction procedures and has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from D. dadantii. The i...

  18. Comparison of three decontamination methods for Mycobacterium bovis isolation

    PubMed Central

    Ambrosio, Simone Rodrigues; de Deus Oliveira, Eugenia Márcia; Rodriguez, Cesar Alejandro Rosales; Ferreira Neto, José Soares; Amaku, Marcos

    2008-01-01

    Sixty samples of tissue fragments with lesions suggestive of tuberculosis from bovine abattoirs, kept in saturated solution of sodium borate, were subjected to four treatments: 4% NaOH (Petroff Method), 12 % H2SO4 and 1.5% HPC (1-Hexadecylpyridinium Chloride) decontamination, and physiological saline solution (control). The HPC method showed the lowest contamination rate (3%) when compared to control (88%, p<0.001), NaOH (33%, p<0.001) and H2SO4 (21.7%, p<0.002). Regarding the isolation success, the HPC method was better (40%) than the control (3%, p<0.001), NaOH (13%, p=0.001) and H2SO4 (1.7%, p<0.001) methods. These results indicate that HPC is an alternative to the Petroff method. PMID:24031209

  19. A Tool Preference Choice Method for RNA Secondary Structure Prediction by SVM with Statistical Tests

    PubMed Central

    Hor, Chiou-Yi; Yang, Chang-Biau; Chang, Chia-Hung; Tseng, Chiou-Ting; Chen, Hung-Hsin

    2013-01-01

    The Prediction of RNA secondary structures has drawn much attention from both biologists and computer scientists. Many useful tools have been developed for this purpose. These tools have their individual strengths and weaknesses. As a result, based on support vector machines (SVM), we propose a tool choice method which integrates three prediction tools: pknotsRG, RNAStructure, and NUPACK. Our method first extracts features from the target RNA sequence, and adopts two information-theoretic feature selection methods for feature ranking. We propose a method to combine feature selection and classifier fusion in an incremental manner. Our test data set contains 720 RNA sequences, where 225 pseudoknotted RNA sequences are obtained from PseudoBase, and 495 nested RNA sequences are obtained from RNA SSTRAND. The method serves as a preprocessing way in analyzing RNA sequences before the RNA secondary structure prediction tools are employed. In addition, the performance of various configurations is subject to statistical tests to examine their significance. The best base-pair accuracy achieved is 75.5%, which is obtained by the proposed incremental method, and is significantly higher than 68.8%, which is associated with the best predictor, pknotsRG. PMID:23641141

  20. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

    PubMed Central

    Reid, Karen E; Olsson, Niclas; Schlosser, James; Peng, Fred; Lund, Steven T

    2006-01-01

    Background Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. Results We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, α-tubulin, β-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. Conclusion In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-α and SAND as the most relevant reference

  1. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    PubMed

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products. PMID:26898909

  2. THE INFLUENCE OF RNA INTEGRITY ON THE DETECTION OF HONEY BEE VIRUSES: MOLECULAR ASSESSMENT OF DIFFERENT SAMPLE STORAGE METHODS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA quality has been considered one of the most critical components for the overall success of RNA-based assays. To ensure accuracy of virus diagnosis by RT-PCR method, it is important to identify an optimal sample storage method that stabilizes RNA and prevents RNA from activities of RNase in int...

  3. Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate.

    PubMed

    Bhagwat, Basdeo; Dickison, Virginia; Ding, Xinlun; Walker, Melanie; Bernardy, Michael; Bouthillier, Michel; Creelman, Alexa; DeYoung, Robyn; Li, Yinzi; Nie, Xianzhou; Wang, Aiming; Xiang, Yu; Sanfaçon, Hélène

    2016-06-01

    In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas. PMID:26984225

  4. Prediction of MicroRNA-Disease Associations Based on Social Network Analysis Methods.

    PubMed

    Zou, Quan; Li, Jinjin; Hong, Qingqi; Lin, Ziyu; Wu, Yun; Shi, Hua; Ju, Ying

    2015-01-01

    MicroRNAs constitute an important class of noncoding, single-stranded, ~22 nucleotide long RNA molecules encoded by endogenous genes. They play an important role in regulating gene transcription and the regulation of normal development. MicroRNAs can be associated with disease; however, only a few microRNA-disease associations have been confirmed by traditional experimental approaches. We introduce two methods to predict microRNA-disease association. The first method, KATZ, focuses on integrating the social network analysis method with machine learning and is based on networks derived from known microRNA-disease associations, disease-disease associations, and microRNA-microRNA associations. The other method, CATAPULT, is a supervised machine learning method. We applied the two methods to 242 known microRNA-disease associations and evaluated their performance using leave-one-out cross-validation and 3-fold cross-validation. Experiments proved that our methods outperformed the state-of-the-art methods. PMID:26273645

  5. Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family.

    PubMed

    Marklewitz, Marco; Zirkel, Florian; Kurth, Andreas; Drosten, Christian; Junglen, Sandra

    2015-06-16

    The evolutionary origins of arboviruses are unknown because their typical dual host tropism is paraphyletic within viral families. Here we studied one of the most diversified and medically relevant RNA virus families, the Bunyaviridae, in which four of five established genera are transmitted by arthropods. We define two cardinally novel bunyavirus groups based on live isolation of 26 viral strains from mosquitoes (Jonchet virus [JONV], eight strains; Ferak virus [FERV], 18 strains). Both viruses were incapable of replicating at vertebrate-typical temperatures but replicated efficiently in insect cells. Replication involved formation of virion-sense RNA (vRNA) and mRNA, including cap-snatching activity. SDS/PAGE, mass spectrometry, and Edman degradation identified translation products corresponding to virion-associated RNA-dependent RNA polymerase protein (RdRp), glycoprotein precursor protein, glycoproteins Gn and Gc, as well as putative nonstructural proteins NSs and NSm. Distinct virion morphologies suggested ancient evolutionary divergence, with bunyavirus-typical morphology for FERV (spheres of 60-120 nm) as opposed to an unusual bimorphology for JONV (tubular virions of 60 × 600 nm and spheres of 80 nm). Both viruses were genetically equidistant from all other bunyaviruses, showing <15% amino acid identity in the RdRp palm domain. Both had different and unique conserved genome termini, as in separate bunyavirus genera. JONV and FERV define two novel sister taxons to the superclade of orthobunyaviruses, tospoviruses, and hantaviruses. Phylogenetic ancestral state reconstruction with probabilistic hypothesis testing suggested ancestral associations with arthropods at deep nodes throughout the bunyavirus tree. Our findings suggest an arthropod origin of bunyaviruses. PMID:26038576

  6. Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family

    PubMed Central

    Marklewitz, Marco; Zirkel, Florian; Kurth, Andreas; Drosten, Christian; Junglen, Sandra

    2015-01-01

    The evolutionary origins of arboviruses are unknown because their typical dual host tropism is paraphyletic within viral families. Here we studied one of the most diversified and medically relevant RNA virus families, the Bunyaviridae, in which four of five established genera are transmitted by arthropods. We define two cardinally novel bunyavirus groups based on live isolation of 26 viral strains from mosquitoes (Jonchet virus [JONV], eight strains; Ferak virus [FERV], 18 strains). Both viruses were incapable of replicating at vertebrate-typical temperatures but replicated efficiently in insect cells. Replication involved formation of virion-sense RNA (vRNA) and mRNA, including cap-snatching activity. SDS/PAGE, mass spectrometry, and Edman degradation identified translation products corresponding to virion-associated RNA-dependent RNA polymerase protein (RdRp), glycoprotein precursor protein, glycoproteins Gn and Gc, as well as putative nonstructural proteins NSs and NSm. Distinct virion morphologies suggested ancient evolutionary divergence, with bunyavirus-typical morphology for FERV (spheres of 60–120 nm) as opposed to an unusual bimorphology for JONV (tubular virions of 60 × 600 nm and spheres of 80 nm). Both viruses were genetically equidistant from all other bunyaviruses, showing <15% amino acid identity in the RdRp palm domain. Both had different and unique conserved genome termini, as in separate bunyavirus genera. JONV and FERV define two novel sister taxons to the superclade of orthobunyaviruses, tospoviruses, and hantaviruses. Phylogenetic ancestral state reconstruction with probabilistic hypothesis testing suggested ancestral associations with arthropods at deep nodes throughout the bunyavirus tree. Our findings suggest an arthropod origin of bunyaviruses. PMID:26038576

  7. Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM).

    PubMed

    Palmirotta, Raffaele; De Marchis, Maria Laura; Ludovici, Giorgia; Leone, Barbara; Savonarola, Annalisa; Ialongo, Cristiano; Spila, Antonella; De Angelis, Francesco; Ferroni, Patrizia; Della-Morte, David; Guadagni, Fiorella

    2012-01-01

    Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).
Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications. PMID:22562396

  8. Isolation and characterization of Saccharomyces cerevisiae mRNA transport-defective (mtr) mutants [published erratum appears in J Cell Biol 1994 Sep;126(6):1627

    PubMed Central

    1994-01-01

    To understand the mechanisms of mRNA transport in eukaryotes, we have isolated Saccharomyces cerevisiae temperature-sensitive (ts) mutants which accumulate poly(A)+ RNA in the nucleus at the restrictive temperature. A total of 21 recessive mutants were isolated and classified into 16 complementation groups. Backcrossed mRNA transport- defective strains from each complementation group have been analyzed. A strain which is ts for heat shock transcription factor was also analyzed since it also shows nuclear accumulation of poly(A)+ RNA at 37 degrees C. At 37 degrees C the mRNA of each mutant is characterized by atypically long polyA tails. Unlike ts pre-mRNA splicing mutants, these strains do not interrupt splicing of pre-mRNA at 37 degrees C; however four strains accumulate oversized RNA polymerase II transcripts. Some show inhibition of rRNA processing and a further subset of these strains is also characterized by inhibition of tRNA maturation. Several strains accumulate nuclear proteins in the cytoplasm when incubated at semipermissive temperature. Remarkably, many strains exhibit nucleolar fragmentation or enlargement at the restrictive temperature. Most strains show dramatic ultrastructural alterations of the nucleoplasm or nuclear membrane. Distinct mutants accumulate poly(A)+ RNA in characteristic patterns in the nucleus. PMID:8045930

  9. Novel 5'/3'RACE Method for Amplification and Determination of Single-Stranded RNAs Through Double-Stranded RNA (dsRNA) Intermediates.

    PubMed

    Pankovics, Péter; Boros, Ákos; Reuter, Gábor

    2015-12-01

    To acquire the full-length sequences and to determine the 5'/3'ends of the RNA genomes and mRNA transcripts using the rapid amplification of cDNA ends (RACE) protocols-via cDNA or mRNA templates-are a great challenge. This 4-steps RNA-based RACE method uses different ways to determine the RNA ends through a double-stranded (ds) RNA intermediate (dsRNA-RACE). In the first step a complementary RNA strand is synthesised by Phi6 RNA replicase enzyme next to the template ssRNA forming a dsRNA intermediate. The following steps include adapter ligation, nucleic acid purification and two classical methods with minor modifications reverse transcription and polymerase chain reaction. The dsRNA-RACE protocol could be used in wide variety of ssRNA (cellular, viral, bacterial, etc.) templates in the field of microbiology and cellular biology and suitable for the amplification of full-length RNAs including the 5'/3'ends. This is a novel, expansively utilizable molecular tool with fewer disadvantages than the existing 5'/3'RACE approaches. PMID:26315976

  10. Tetracycline-Resistant Clinical Helicobacter pylori Isolates with and without Mutations in 16S rRNA-Encoding Genes

    PubMed Central

    Wu, Jeng Yih; Kim, Jae J.; Reddy, Rita; Wang, W. M.; Graham, David Y.; Kwon, Dong H.

    2005-01-01

    Tetracycline-resistant Helicobacter pylori strains have been increasingly reported worldwide. However, only a small number of tetracycline-resistant strains have been studied with regard to possible mechanisms of resistance and those studies have focused on mutations in the tetracycline binding sites of 16S rRNA-encoding genes. We here report studies of 41 tetracycline-resistant H. pylori strains (tetracycline MICs, 4 to 32 μg/ml) from North America (n = 12) and from East Asia (n = 29). DNA sequence analyses of 16S rRNA-encoding genes revealed that 22 (54%) of the resistant isolates carried one of five different single-nucleotide substitutions (CGA, GGA, TGA, AGC, or AGT) at the putative tetracycline binding site (AGA965-967). Single-nucleotide substitutions were associated with reduced ribosomal binding and with slightly increased tetracycline MICs (1 to 2 μg/ml). The 19 tetracycline-resistant isolates with no detectable mutations in the tetracycline binding site had normal tetracycline-ribosome binding. All tetracycline-resistant isolates, including those with and those without mutations in the tetracycline binding site, showed decreased accumulation of tetracycline. These results suggest that tetracycline resistance is multifactorial, involving alterations both in ribosomal binding and in membrane permeability. PMID:15673736

  11. Robust computational reconstitution – a new method for the comparative analysis of gene expression in tissues and isolated cell fractions

    PubMed Central

    Hoffmann, Martin; Pohlers, Dirk; Koczan, Dirk; Thiesen, Hans-Jürgen; Wölfl, Stefan; Kinne, Raimund W

    2006-01-01

    Background Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis. Results The expression profiles of the whole tissues were compared to computationally reconstituted expression profiles that combine the expression profiles of the isolated cell fractions (macrophages, fibroblasts, and non-adherent cells) according to their relative mRNA proportions in the tissue. The mRNA proportions were determined by trimmed robust regression using only the most robustly-expressed genes (1/3 to 1/2 of all measured genes), i.e. those showing the most similar expression in tissue and isolated cell fractions. The relative mRNA proportions were determined using several different chip evaluation methods, among which the MAS 5.0 signal algorithm appeared to be most robust. The computed mRNA proportions agreed well with the cell proportions determined by immunohistochemistry except for a minor number of outliers. Genes that were either regulated (i.e. differentially-expressed in tissue and isolated cell fractions) or robustly-expressed in all patients were identified using different test statistics. Conclusion Robust Computational Reconstitution uses an intermediate number of robustly-expressed genes to estimate the relative mRNA proportions. This avoids both the exclusive dependence on the robust expression of individual, highly cell type-specific marker genes and the bias towards an equal distribution upon inclusion of all genes for computation. PMID:16889662

  12. Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.

    PubMed

    Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica; Court, Carolyn; Chen, Shuo; Court, Donald L; Strathern, Jeffrey; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-25

    Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. PMID:23223236

  13. Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli*

    PubMed Central

    Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica; Court, Carolyn; Chen, Shuo; Court, Donald L.; Strathern, Jeffrey; Jin, Ding Jun; Kashlev, Mikhail

    2013-01-01

    Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity. PMID:23223236

  14. Alternative methods for the efficient construction of short hairpin RNA expression vectors.

    PubMed

    Xu, Kun; Zhang, Tingting; Guo, Lijun; Xin, Ying; Zhang, Long; Zhang, Zhiying

    2015-06-01

    Short hairpin RNA (shRNA)-mediated RNA interference has become a basic technique in modern molecular biology and biochemistry for studying gene function and biological pathways. Here, we report two alternative and efficient methods to construct shRNA expression vectors based respectively on multiple-step sequential PCR and primer extension-homologous recombination (PE-HR). Neither method requires synthesizing long oligonucleotides containing hairpin sequences as used in traditional approaches. The hairpin sequences may produce mutations during oligo synthesis, pose problems in annealing, and lead to inefficient cloning. The PE-HR method further provides rapid and economical construction of shRNA expression vectors without needing the ligation procedure. PMID:25794926

  15. α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

    PubMed Central

    Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

    1981-01-01

    [3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

  16. A method for clustering of miRNA sequences using fragmented programming.

    PubMed

    Ivashchenko, Anatoly; Pyrkova, Anna; Niyazova, Raigul

    2016-01-01

    Clustering of miRNA sequences is an important problem in molecular genetics associated cellular biology. Thousands of such sequences are known today through advancement in sophisticated molecular tools, sequencing techniques, computational resources and rule based mathematical models. Analysis of such large-scale miRNA sequences for inferring patterns towards deducing cellular function is a great challenge in modern molecular biology. Therefore, it is of interest to develop mathematical models specific for miRNA sequences. The process is to group (cluster) such miRNA sequences using well-defined known features. We describe a method for clustering of miRNA sequences using fragmented programming. Subsequently, we illustrated the utility of the model using a dendrogram (a tree diagram) for publically known A.thaliana miRNA nucleotide sequences towards the inference of observed conserved patterns. PMID:27212839

  17. Easier, Better, Faster, Stronger: Improved Methods for RNA-Protein Interaction Studies.

    PubMed

    Haque, Nazmul; Hogg, J Robert

    2016-06-01

    The RNA field has been revolutionized by methods that allow genome-scale identification of RNA-protein interaction sites. Two reports now introduce more efficient approaches, opening the technology to wider adoption (Van Nostrand et al., 2016; Zarnegar et al., 2016). PMID:27259196

  18. [Isolation and in vitro translation of polysomes and the RNA matrix from gibberellic acid-treated etiolated dwarf pea shoots].

    PubMed

    Kilev, S N; Evtushenko, E V; Chekurov, V M; Mertvetsov, N P

    1983-08-01

    The effect of gibberellic acid (GA) on the size of polysomes (PS) and on the specific translation activity of epicotyl PS of two dwarf pea varieties was studied. It was shown that GA does not significantly alter the specific translation activity of PS and of mRNA isolated from dwarf pea epicotyl PS. Electrophoretic separation of the polypeptides synthesized in a cell-free protein-synthesizing system in PS of control and GA-treated shoots revealed no differences between them. Some minor qualitative and quantitative differences in the protein composition of the cytoplasm of control and hormone-treated shoots were found. Possible influence of GA on the composition of a definite class of mRNA and on posttranslational processing of plant proteins is discussed. PMID:6194827

  19. Isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mRNA.

    PubMed

    Jha, Sujata S; Komar, Anton A

    2012-01-01

    The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message(for review see 1). However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates(1). Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others(2). Codon bias is organism as well as tissue specific(2,3). Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs(4). Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes(5-8). These pause sites can have functional impact on the protein expression, mRNA stability and protein folding(for review see 9). Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding(1,7,10,11). To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation. Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems(6-8). This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the

  20. Differential amplicons (ΔAmp)—a new molecular method to assess RNA integrity

    PubMed Central

    Björkman, J.; Švec, D.; Lott, E.; Kubista, M.; Sjöback, R.

    2015-01-01

    Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow. PMID:27077042

  1. Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-dilution Method

    PubMed Central

    Fu, Lijuan; Amato, Nicolas J.; Wang, Pengcheng; McGowan, Sara J.; Niedernhofer, Laura J.; Wang, Yinsheng

    2016-01-01

    The rising interest in understanding the functions, regulation and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Mono-methylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2′-O-methylcytidine (Cm), N6-methyladenosine (m6A) and 2′-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of the four methylated nucleosides are tissue-specific. In addition, the 2′-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m5C and m6A) in total RNA isolated from mouse brain, pancreas and spleen, but not mouse heart. We also found that the levels of m5C, Cm and Am are significantly lower (by 6.5-43 fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6 fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications. PMID:26158405

  2. Molecular Methods for Identification of Acinetobacter Species by Partial Sequencing of the rpoB and 16S rRNA Genes

    PubMed Central

    Khosravi, Azar Dokht; Shahraki, Abdolrazagh Hashemi; Heidarieh, Parvin; Sheikhi, Nasrin

    2015-01-01

    Background Acinetobacter spp. is a diverse group of Gram-negative bacteria which are ubiquitous in soil and water, and an important cause of nosocomial infections. The purpose of this study was to identify a collection of Acinetobacter spp. clinical isolates accurately and to investigate their antibiotic susceptibility patterns. Materials and Methods A total of 197 non-duplicate clinical isolates of Acinetobacter spp. isolates identified using conventional biochemical tests. The molecular technique of PCR-RFLP and sequence analysis of rpoB and 16S rRNA genes was applied for species identification. Antimicrobial susceptibility test was performed with a disk diffusion assay. Results Based on 16S rRNA and rpoB genes analysis separately, most of clinical isolates can be identified with high bootstrap values. However, the identity of the isolate 555T was uncertain due to high similarity of A. grimontii and A. junii. Identification by concatenation of 16S rRNA and rpoB confirmed the identity of clinical isolates of Acenitobacer to species level confidently. Accordingly, the isolate 555T assigned as A. grimontii due to 100% similarity to A. grimontii. Moreover, this isolate showed 98.64% to A. junii. Besides, the identity of the isolates 218T and 364T was confirmed as Genomic species 3 and A. calcoaceticus respectively. So, the majority of Acinetobacter spp. isolates, were identified as: A. baumannii (131 isolates, 66%), A. calcoaceticus (9 isolates, 4.5%), and A. genomosp 16 (8 isolates, 4%). The rest of identified species showed the lower frequencies. In susceptibility test, 105 isolates (53%), presented high antibiotic resistance of 90% to ceftriaxone, piperacillin, piperacillin tazobactam, amikacin, and 81% to ciprofloxacin. Conclusion Sequence analysis of the 16S rRNA and rpoB spacer simultaneously was able to do identification of Acinetobacter spp. to species level. A.baumannii was identified as the most prevalent species with high antibiotic resistance. Other

  3. Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda

    PubMed Central

    Bleckmann, Maren; Fritz, Markus H.-Y.; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

    2015-01-01

    The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins-especially those entering the secretory pathway- provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially. PMID:26263512

  4. Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

    PubMed Central

    Moreira, João Luiz S; Mota, Rodrigo M; Horta, Maria F; Teixeira, Santuza MR; Neumann, Elisabeth; Nicoli, Jacques R; Nunes, Álvaro C

    2005-01-01

    Background The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. Results Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products. Conclusion Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies. PMID:15788104

  5. Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

    PubMed

    Sempere, Raquel N; Gómez-Aix, Cristina; Ruíz-Ramón, Fabiola; Gómez, Pedro; Hasiów-Jaroszewska, Beata; Sánchez-Pina, María Amelia; Aranda, Miguel A

    2016-04-01

    Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions. PMID:26667188

  6. Rapid alkaline extraction method for the isolation of plasmid DNA

    SciTech Connect

    Birnboim, H.C.

    1983-01-01

    Plasmids are double-stranded circular DNA molecules that have the property of self-replication, independent of chromosomal DNA. Although the presence of a plasmid in a bacterial cell may be detected genetically as a change in phenotype, often it is necessary to isolate plasmid DNA for molecular studies, such as size determination, restriction enzyme mapping, and nucleotide sequencing, or for the construction of new hybrid plasmids. The degree of purification required will depend upon the intended use. Less purified plasmid DNA is often satisfactory for recombinant DNA studies, and a large number of shorter and simpler methods have been developed. This chapter describes one such method that uses an alkaline extraction step. It is rapid enough to be used as a screening method, permitting 50-100 or more samples to be extracted in a few hours. The DNA is sufficiently pure to be digestible by restriction enzymes, an important advantage for screening. A preparative version that allows isolation of larger quantities of more highly purified material is also described.

  7. Murine mammary stem/progenitor cell isolation: Different method matters?

    PubMed

    Gao, Hui; Dong, Qiaoxiang; Chen, Yuanhong; Zhang, Fuchuang; Wu, Anqi; Shi, Yuanshuo; Bandyopadhyay, Abhik; Daniel, Benjamin J; Huang, Changjiang; Sun, Lu-Zhe

    2016-01-01

    Murine mammary stem/progenitor cell isolation has been routinely used in many laboratories, yet direct comparison among different methods is lacking. In this study, we compared two frequently used digestion methods and three sets of frequently used surface markers for their efficiency in enriching mammary stem and progenitor cells in two commonly used mouse strains, C57BL/6J and FVB. Our findings revealed that the slow overnight digestion method using gentle collagenase/hyaluronidase could be easily adopted and yielded reliable and consistent results in different batches of animals. In contrast, the different fast digestion protocols, as described in published studies, yielded high percent of non-epithelial cells with very few basal epithelial cells liberated in our hands. The three sets of markers tested in our hands reveal rather equally efficiency in separating luminal and basal cells if same fluorochrome conjugations were used. However, the tendency of non-epithelial cell inclusion in the basal cell gate was highest in samples profiled by CD24/CD29 and lowest in samples profiled by CD49f/EpCAM, this is especially true in mammary cells isolated from C57BL/6J mice. This finding will have significant implication when sorted basal cells are used for subsequent gene expression analysis. PMID:26933638

  8. Can we observe changes in mRNA “state”? Overview of methods to study mRNA interactions with regulatory proteins relevant in cancer related processes

    PubMed Central

    Zurla, C.; Jung, J.; Santangelo, P. J.

    2015-01-01

    RNA binding proteins (RBP) regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNA) through their interactions with specific cis-acting elements within target RNAs. Post-transcriptional regulatory mechanisms are directly involved in the control of the immune response and stress response and their alterations play a crucial role in cancer related processes. In this review, we discuss mRNAs and RNA binding proteins relevant to tumorigenesis, current methodologies for detecting RNA interactions, and last, we describe a novel method to detect such interactions, which combines peptide modified, RNA imaging probes (FMTRIPs) with proximity ligation (PLA) and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein–mRNA interactions with single-interaction sensitivity in situ. PMID:26605378

  9. Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

    PubMed

    Oliveira, R R; Viana, A J C; Reátegui, A C E; Vincentz, M G A

    2015-01-01

    Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses. PMID:26782533

  10. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    NASA Astrophysics Data System (ADS)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  11. Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry

    PubMed Central

    Mooers, Blaine H. M.

    2016-01-01

    Using direct methods starting from random phases, the crystal structure of a 32-base-pair RNA (675 non-H RNA atoms in the asymmetric unit) was determined using only the native diffraction data (resolution limit 1.05 Å) and the computer program SIR2014. The almost three helical turns of the RNA in the asymmetric unit introduced partial or imperfect translational pseudosymmetry (TPS) that modulated the intensities when averaged by the l Miller indices but still escaped automated detection. Almost six times as many random phase sets had to be tested on average to reach a correct structure compared with a similar-sized RNA hairpin (27 nucleotides, 580 non-H RNA atoms) without TPS. More sensitive methods are needed for the automated detection of partial TPS. PMID:27050127

  12. Fungal endophytes in potato roots studied by traditional isolation and cultivation-independent DNA-based methods.

    PubMed

    Götz, Monika; Nirenberg, Helgard; Krause, Sibylle; Wolters, Heike; Draeger, Siegfried; Buchner, Arno; Lottmann, Jana; Berg, Gabriele; Smalla, Kornelia

    2006-12-01

    The composition and relative abundance of endophytic fungi in roots of field-grown transgenic T4-lysozyme producing potatoes and the parental line were assessed by classical isolation from root segments and cultivation-independent techniques to test the hypothesis that endophytic fungi are affected by T4-lysozyme. Fungi were isolated from the majority of root segments of both lines and at least 63 morphological groups were obtained with Verticillium dahliae, Cylindrocarpon destructans, Colletotrichum coccodes and Plectosporium tabacinum as the most frequently isolated species. Dominant bands in the fungal fingerprints obtained by denaturing gradient gel electrophoresis analysis of 18S rRNA gene fragments amplified from total community DNA corresponded to the electrophoretic mobility of the 18S rRNA gene fragments of the three most abundant fungal isolates, V. dahliae, C. destructans and Col. coccodes, but not to P. tabacinum. The assignment of the bands to these isolates was confirmed for V. dahliae and Col. coccodes by sequencing of clones. Verticillium dahliae was the most abundant endophytic fungus in the roots of healthy potato plants. Differences in the relative abundance of endophytic fungi colonizing the roots of T4-lysozyme producing potatoes and the parental line could be detected by both methods. PMID:17117985

  13. PCR amplification of rRNA intergenic spacer regions as a method for epidemiologic typing of Clostridium difficile.

    PubMed Central

    Cartwright, C P; Stock, F; Beekmann, S E; Williams, E C; Gill, V J

    1995-01-01

    From January to March 1993, a suspected outbreak of antibiotic-associated diarrhea occurred on a pediatric oncology ward of the Clinical Center Hospital at the National Institutes of Health. Isolates of Clostridium difficile obtained from six patients implicated in this outbreak were typed by both PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) and restriction endonuclease analysis of genomic DNA. Comparable results were obtained with both methods; five of the six patients were infected with the same strain of C. difficile. Subsequent analysis of 102 C. difficile isolates obtained from symptomatic patients throughout the Clinical Center revealed the existence of 41 distinct and reproducible PCR ribotypes. These data suggest that PCR ribotyping provides a discriminatory, reproducible, and simple alternative to conventional molecular approaches for typing strains of C. difficile. PMID:7699038

  14. Sensitive and specific miRNA detection method using SplintR Ligase.

    PubMed

    Jin, Jingmin; Vaud, Sophie; Zhelkovsky, Alexander M; Posfai, Janos; McReynolds, Larry A

    2016-07-27

    We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection. PMID:27154271

  15. Crumple: a method for complete enumeration of all possible pseudoknot-free RNA secondary structures.

    PubMed

    Bleckley, Samuel; Stone, Jonathan W; Schroeder, Susan J

    2012-01-01

    The diverse landscape of RNA conformational space includes many canyons and crevices that are distant from the lowest minimum free energy valley and remain unexplored by traditional RNA structure prediction methods. A complete description of the entire RNA folding landscape can facilitate identification of biologically important conformations. The Crumple algorithm rapidly enumerates all possible non-pseudoknotted structures for an RNA sequence without consideration of thermodynamics while filtering the output with experimental data. The Crumple algorithm provides an alternative approach to traditional free energy minimization programs for RNA secondary structure prediction. A complete computation of all non-pseudoknotted secondary structures can reveal structures that would not be predicted by methods that sample the RNA folding landscape based on thermodynamic predictions. The free energy minimization approach is often successful but is limited by not considering RNA tertiary and protein interactions and the possibility that kinetics rather than thermodynamics determines the functional RNA fold. Efficient parallel computing and filters based on experimental data make practical the complete enumeration of all non-pseudoknotted structures. Efficient parallel computing for Crumple is implemented in a ring graph approach. Filters for experimental data include constraints from chemical probing of solvent accessibility, enzymatic cleavage of paired or unpaired nucleotides, phylogenetic covariation, and the minimum number and lengths of helices determined from crystallography or cryo-electron microscopy. The minimum number and length of helices has a significant effect on reducing conformational space. Pairing constraints reduce conformational space more than single nucleotide constraints. Examples with Alfalfa Mosaic Virus RNA and Trypanosome brucei guide RNA demonstrate the importance of evaluating all possible structures when pseduoknots, RNA-protein interactions

  16. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  17. Pouch Method for the Isolation and Enumeration of Clostridia

    PubMed Central

    Bladel, B. O.; Greenberg, Richard A.

    1965-01-01

    An anaerobic film-pouch method has been developed for the isolation and enumeration of clostridia. Fabrication of the pouch is described. Counts of spore suspensions of Putrefactive Anaerobe 3679 and of Clostridium botulinum strains 41-B and 33-A in pouches were compared with those obtained by anaerobic-jar and agar-deep techniques. Statistical analysis revealed a significant difference in favor of the pouch over the tube and anaerobic-jar methods. Tests performed with C. welchii, both in spore suspension and added to chicken pot pie in culture form, also demonstrated the pouch to be at least as proficient as the other, more cumbersome, techniques. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:14325895

  18. Method for localizing and isolating an errant process step

    DOEpatents

    Tobin, Jr., Kenneth W.; Karnowski, Thomas P.; Ferrell, Regina K.

    2003-01-01

    A method for localizing and isolating an errant process includes the steps of retrieving from a defect image database a selection of images each image having image content similar to image content extracted from a query image depicting a defect, each image in the selection having corresponding defect characterization data. A conditional probability distribution of the defect having occurred in a particular process step is derived from the defect characterization data. A process step as a highest probable source of the defect according to the derived conditional probability distribution is then identified. A method for process step defect identification includes the steps of characterizing anomalies in a product, the anomalies detected by an imaging system. A query image of a product defect is then acquired. A particular characterized anomaly is then correlated with the query image. An errant process step is then associated with the correlated image.

  19. [The method of the isolated reconstruction by gastropancreatoduodenal resection].

    PubMed

    Shchepotin, I B; Vasil'ev, O V; Lukashenko, A V; Rozumiĭ, D A; Priĭmak, V V

    2011-01-01

    The modification of the reconstructive stage of gastropancreatoduodenal resection aims to increase the security of the pancreatojejunoanastomosis by minimizing the impact of such aggressive substances as bile and pancreatic juice. The modification represents the isolated pancreatojejunoanastomosis on the Roux-en-Y intestinal loop and gastro- and hepaticojejunoanastomoses on the second intestinal loop, separated with the use of the stub. Thus, the method allows the separate passage of pancreatic juice, bile and gastric contents, excluding their impact on other anastomoses. The described modification was performed in 6 patients. There were no cases of the anastomotic insufficiency. The mean hospital stay was 10,5 days. Thus. The method proved to be effective and safe, providing good initial results. PMID:22334901

  20. Isolation of RNA striped bass Monrone saxatilis spermatozoa: Implications for teleost male fertility and beyond?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vertebrate sperm has been shown to provide more than paternal genomic material to the oocyte. For example, specific transcripts have been identified in bull sperm associated with fertility and with motility in boar sperm. Very little is currently known about functional, residual RNA in spermatozoa a...

  1. Specific isolation of RNA from the grape powdery mildew pathogen Erysiphe necator, an epiphytic, obligate parasite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA expression profiling of obligately parasitic plant microbes is hampered by the requisite interaction of host and parasite. For superficial pathogens like grape powdery mildew as well as for epiphytic saprophytes, growth along the outside surface of the plant allows separation from the host and ...

  2. Development of a low-cost detection method for miRNA microarray.

    PubMed

    Li, Wei; Zhao, Botao; Jin, Youxin; Ruan, Kangcheng

    2010-04-01

    MicroRNA (miRNA) microarray is a powerful tool to explore the expression profiling of miRNA. The current detection method used in miRNA microarray is mainly fluorescence based, which usually requires costly detection system such as laser confocal scanner of tens of thousands of dollars. Recently, we developed a low-cost yet sensitive detection method for miRNA microarray based on enzyme-linked assay. In this approach, the biotinylated miRNAs were captured by the corresponding oligonucleotide probes immobilized on microarray slide; and then the biotinylated miRNAs would capture streptavidin-conjugated alkaline phosphatase. A purple-black precipitation on each biotinylated miRNA spot was produced by the enzyme catalytic reaction. It could be easily detected by a charge-coupled device digital camera mounted on a microscope, which lowers the detection cost more than 100 fold compared with that of fluorescence method. Our data showed that signal intensity of the spot correlates well with the biotinylated miRNA concentration and the detection limit for miRNAs is at least 0.4 fmol and the detection dynamic range spans about 2.5 orders of magnitude, which is comparable to that of fluorescence method. PMID:20383469

  3. Niemeyer Virus: A New Mimivirus Group A Isolate Harboring a Set of Duplicated Aminoacyl-tRNA Synthetase Genes

    PubMed Central

    Boratto, Paulo V. M.; Arantes, Thalita S.; Silva, Lorena C. F.; Assis, Felipe L.; Kroon, Erna G.; La Scola, Bernard; Abrahão, Jônatas S.

    2015-01-01

    It is well recognized that gene duplication/acquisition is a key factor for molecular evolution, being directly related to the emergence of new genetic variants. The importance of such phenomena can also be expanded to the viral world, with impacts on viral fitness and environmental adaptations. In this work we describe the isolation and characterization of Niemeyer virus, a new mimivirus isolate obtained from water samples of an urban lake in Brazil. Genomic data showed that Niemeyer harbors duplicated copies of three of its four aminoacyl-tRNA synthetase genes (cysteinyl, methionyl, and tyrosyl RS). Gene expression analysis showed that such duplications allowed significantly increased expression of methionyl and tyrosyl aaRS mRNA by Niemeyer in comparison to APMV. Remarkably, phylogenetic data revealed that Niemeyer duplicated gene pairs are different, each one clustering with a different group of mimivirus strains. Taken together, our results raise new questions about the origins and selective pressures involving events of aaRS gain and loss among mimiviruses. PMID:26635738

  4. Improvement on the extraction method of RNA in mites and its quality test.

    PubMed

    Zhao, YaE; Hu, Li; Yang, Yuan Jun; Niu, Dong Ling; Wang, Rui Ling; Li, Wen Hao; Ma, Si Jia; Cheng, Juan

    2016-02-01

    To solve the long-existing difficult problems in extracting RNA and constructing a complementary DNA (cDNA) library for trace mites, we conducted a further comparative experiment among three RNA extraction methods (TRIzol method, Omega method, and Azanno method) based on our previous attempts at the construction of cDNA library of mites, with Psoroptes cuniculi still used as the experimental subject. By subsequently decreasing the number of mites, the least number of mites needed for RNA extraction of each method were found by criteria of completeness, concentration, and purity of the extracted RNA. Specific primers were designed according to the allergen Pso c1, Pso c2, and Actin gene sequences of Psoroptes to test the reliability of cDNA library. The results showed that Azanno method needed only 10 mites with sensitivity 204 times higher than previously used TRIzol method and 20 times higher than Omega method; clear RNA band was detected by agarose gel electrophoresis; and ultraviolet spectrophotometer determination showed that RNA concentration, 260/280, and 260/230 were in the range of 102 to 166 ng/μl, 1.83 to 1.99, and 1.49 to 1.72, respectively. Finally, specific primers detection showed that the amplified sequences had 98.33, 98.19, and 99.52% identities with those of P. cuniculi or Psoroptes ovis in GenBank, respectively, indicating that the cDNA library constructed using 10 mites was successful and it could meet the requirements for molecular biology research. Therefore, we concluded that Azanno method was more effective than TRIzol method and Omega method in RNA extraction and cDNA library construction of trace mites. PMID:26545909

  5. ADVANCED SEISMIC BASE ISOLATION METHODS FOR MODULAR REACTORS

    SciTech Connect

    E. Blanford; E. Keldrauk; M. Laufer; M. Mieler; J. Wei; B. Stojadinovic; P.F. Peterson

    2010-09-20

    Advanced technologies for structural design and construction have the potential for major impact not only on nuclear power plant construction time and cost, but also on the design process and on the safety, security and reliability of next generation of nuclear power plants. In future Generation IV (Gen IV) reactors, structural and seismic design should be much more closely integrated with the design of nuclear and industrial safety systems, physical security systems, and international safeguards systems. Overall reliability will be increased, through the use of replaceable and modular equipment, and through design to facilitate on-line monitoring, in-service inspection, maintenance, replacement, and decommissioning. Economics will also receive high design priority, through integrated engineering efforts to optimize building arrangements to minimize building heights and footprints. Finally, the licensing approach will be transformed by becoming increasingly performance based and technology neutral, using best-estimate simulation methods with uncertainty and margin quantification. In this context, two structural engineering technologies, seismic base isolation and modular steel-plate/concrete composite structural walls, are investigated. These technologies have major potential to (1) enable standardized reactor designs to be deployed across a wider range of sites, (2) reduce the impact of uncertainties related to site-specific seismic conditions, and (3) alleviate reactor equipment qualification requirements. For Gen IV reactors the potential for deliberate crashes of large aircraft must also be considered in design. This report concludes that base-isolated structures should be decoupled from the reactor external event exclusion system. As an example, a scoping analysis is performed for a rectangular, decoupled external event shell designed as a grillage. This report also reviews modular construction technology, particularly steel-plate/concrete construction using

  6. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods.

    PubMed

    Greening, David W; Xu, Rong; Ji, Hong; Tauro, Bow J; Simpson, Richard J

    2015-01-01

    Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers). PMID:25820723

  7. A comprehensive comparison of general RNA–RNA interaction prediction methods

    PubMed Central

    Lai, Daniel; Meyer, Irmtraud M.

    2016-01-01

    RNA–RNA interactions are fast emerging as a major functional component in many newly discovered non-coding RNAs. Basepairing is believed to be a major contributor to the stability of these intermolecular interactions, much like intramolecular basepairs formed in RNA secondary structure. As such, using algorithms similar to those for predicting RNA secondary structure, computational methods have been recently developed for the prediction of RNA–RNA interactions. We provide the first comprehensive comparison comprising 14 methods that predict general intermolecular basepairs. To evaluate these, we compile an extensive data set of 54 experimentally confirmed fungal snoRNA–rRNA interactions and 102 bacterial sRNA–mRNA interactions. We test the performance accuracy of all methods, evaluating the effects of tool settings, sequence length, and multiple sequence alignment usage and quality. Our results show that—unlike for RNA secondary structure prediction—the overall best performing tools are non-comparative energy-based tools utilizing accessibility information that predict short interactions on this data set. Furthermore, we find that maintaining high accuracy across biologically different data sets and increasing input lengths remains a huge challenge, causing implications for de novo transcriptome-wide searches. Finally, we make our interaction data set publicly available for future development and benchmarking efforts. PMID:26673718

  8. Establishing reliable miRNA-cancer association network based on text-mining method.

    PubMed

    Li, Lun; Hu, Xingchi; Yang, Zhaowan; Jia, Zhenyu; Fang, Ming; Zhang, Libin; Zhou, Yanhong

    2014-01-01

    Associating microRNAs (miRNAs) with cancers is an important step of understanding the mechanisms of cancer pathogenesis and finding novel biomarkers for cancer therapies. In this study, we constructed a miRNA-cancer association network (miCancerna) based on more than 1,000 miRNA-cancer associations detected from millions of abstracts with the text-mining method, including 226 miRNA families and 20 common cancers. We further prioritized cancer-related miRNAs at the network level with the random-walk algorithm, achieving a relatively higher performance than previous miRNA disease networks. Finally, we examined the top 5 candidate miRNAs for each kind of cancer and found that 71% of them are confirmed experimentally. miCancerna would be an alternative resource for the cancer-related miRNA identification. PMID:24895499

  9. Simple method of isolating humic acids from organic soils

    NASA Astrophysics Data System (ADS)

    Ahmed, O. H.; Susilawati, K.; Nik Muhamad, A. B.; Khanif, M. Y.

    2009-04-01

    Humic substances particularly humic acids (HA) play a major role in soil conditioning e.g. erosion control, soil cation exchange capacity, complexation of heavy metal ions and pesticides, carbon and nitrogen cycles, plant growth and reduction of ammonia volatilization from urea. Humified substances such as coal, composts, and peat soils have substantial amounts of HA but the isolation of these acids is expensive, laborious, and time consuming. Factors that affect the quality and yield of HA isolated from these materials include extraction, fractionation, and purification periods. This work developed a simple, rapid, and cost effective method of isolating HA from peat soils. There was a quadratic relationship between extraction period and HA yield. Optimum extraction period was estimated at 4 h instead of the usual range of 12 to 48 h. There was no relationship between fractionation period and HA yield. As such 2 h instead of the usual range of 12 to 24 h fractionation period could be considered optimum. Low ash content (5%), remarkable reduction in K, coupled with the fact that organic C, E4/E6, carboxylic COOH, phenolic OH, and total acidity values of the HA were consistent with those reported by other authors suggest that the HA dealt with were free from mineral matter. This was possible because the distilled water used to purify the HA served as Bronsted-Lowry acid during the purification process of the HA. Optimum purification period using distilled waster was 1 h instead of the usual range of 1 and 7 days (uses HF and HCl and dialysis). Humic acids could be isolated from tropical peat soils within 7 h (i.e. 4 h extraction, 2 h fractionation, and 1 h purification) instead of the existing period of 2 and 7 days. This could facilitate the idea of producing organic fertilizers such as ammonium-humate and potassium-humate from humified substances since techniques devised in this study did not alter the true nature of the HA. Besides, the technique is rapid, simple

  10. A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    PubMed Central

    Arakawa, Hiroshi

    2016-01-01

    The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species. PMID:27574704

  11. A MOTIF-BASED METHOD FOR PREDICTING INTERFACIAL RESIDUES IN BOTH THE RNA AND PROTEIN COMPONENTS OF PROTEIN-RNA COMPLEXES

    PubMed Central

    MANN, CARLA M.; DOBBS, DRENA

    2015-01-01

    Efforts to predict interfacial residues in protein-RNA complexes have largely focused on predicting RNA-binding residues in proteins. Computational methods for predicting protein-binding residues in RNA sequences, however, are a problem that has received relatively little attention to date. Although the value of sequence motifs for classifying and annotating protein sequences is well established, sequence motifs have not been widely applied to predicting interfacial residues in macromolecular complexes. Here, we propose a novel sequence motif-based method for “partner-specific” interfacial residue prediction. Given a specific protein-RNA pair, the goal is to simultaneously predict RNA binding residues in the protein sequence and protein-binding residues in the RNA sequence. In 5-fold cross validation experiments, our method, PS-PRIP, achieved 92% Specificity and 61% Sensitivity, with a Matthews correlation coefficient (MCC) of 0.58 in predicting RNA-binding sites in proteins. The method achieved 69% Specificity and 75% Sensitivity, but with a low MCC of 0.13 in predicting protein binding sites in RNAs. Similar performance results were obtained when PS-PRIP was tested on two independent “blind” datasets of experimentally validated protein-RNA interactions, suggesting the method should be widely applicable and valuable for identifying potential interfacial residues in protein-RNA complexes for which structural information is not available. The PS-PRIP webserver and datasets are available at: http://pridb.gdcb.iastate.edu/PSPRIP/. PMID:26776208

  12. Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

    PubMed Central

    Munroe, S H; Duthie, R S

    1986-01-01

    The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing. Images PMID:3024107

  13. Identification of Cryptosporidium isolates from cockatiels by direct sequencing of the PCR-amplified small subunit ribosomal RNA gene.

    PubMed

    Abe, Niichiro; Iseki, Motohiro

    2004-04-01

    Cryptosporidium is a significant pathogen in humans and animals. Cases of infection by C. meleagridis or C. baileyi with zoonotic potential have also been reported in domestic birds; and recent studies indicate the presence of new host-adapted species or genotype in birds. Therefore, accurately identifying isolates is important for understanding the epizootiology of Cryptosporidium infection in birds and for the control of human cryptosporidiosis. Cryptosporidium has been detected in cockatiels, but the species or genotype of isolates remains unclear because identification was performed using conventional microscopy. We report herein the species or genotype of isolates from two cockatiels distinguished by a PCR-based diagnostic method. The isolates were found to be C. meleagridis and C. baileyi, respectively. This study documents the first discovery of C. meleagridis and C. baileyi in cockatiels and suggests that pet birds may play an important role in the epidemiology of cryptosporidiosis. PMID:14999470

  14. Method for isolating two aquifers in a single borehole

    DOEpatents

    Burklund, Patrick W.

    1985-10-22

    A method for isolating and individually instrumenting separate aquifers within a single borehole. A borehole is first drilled from the ground surface, through an upper aquifer, and into a separating confining bed. A casing, having upper and lower sections separated by a coupling collar, is lowered into the borehole. The borehole is grouted in the vicinity of the lower section of the casing. A borehole is then drilled through the grout plug and into a lower aquifer. After the lower aquifer is instrumented, the borehole is grouted back into the lower portion of the casing. Then the upper section of the casing is unscrewed via the coupling collar and removed from the borehole. Finally, instrumentation is added to the upper aquifer and the borehole is appropriately grouted. The coupling collar is designed to have upper right-hand screw threads and lower left-hand screw thread, whereby the sections of the casing can be readily separated.

  15. Method for isolating two aquifers in a single borehole

    DOEpatents

    Burklund, P.W.

    1984-01-20

    A method for isolating and individually instrumenting separate aquifers within a single borehole is disclosed. A borehole is first drilled from the ground surface, through an upper aquifer, and into a separating confining bed. A casing, having upper and lower sections separated by a coupling collar, is lowered into the borehole. The borehole is grouted in the vicinity of the lower section of the casing. A borehole is then drilled through the grout plug and into a lower aquifer. After the lower aquifer is instrumented, the borehole is grouted back into the lower portion of the casing. Then the upper section of the casing is unscrewed via the coupling collar and removed from the borehole. Finally, instrumentation is added to the upper aquifer and the borehole is appropriately grouted. The coupling collar is designed to have upper right-hand screw threads and lower left-hand screw thread, whereby the sections of the casing can be readily separated.

  16. Urinary extracellular microvesicles: isolation methods and prospects for urinary proteome.

    PubMed

    Wang, Danqi; Sun, Wei

    2014-08-01

    Extracellular microvesicles (EVs) are membranous vesicles, which are released from diverse cells. These EVs have also been found in a wide range of body fluids. The cargo of EVs, including proteins, lipids, carbohydrates, and nucleic acids, can be stably preserved in EVs. Researchers have found that EVs can mediate intercellular communication by shuttling the cargo components. Therefore, EVs can be used for the identification of disease-specific biomarkers. As one class of EVs, urinary exosomes can reflect the status of the renal system. Moreover, urinary exosome analysis can minimize the interference of high abundant proteins in the whole urine sample. Therefore, urinary exosomes have gained much attention in recent years. In this review, we present a comprehensive summary of urinary exosome studies in recent years, including collection, storage, and isolation methods. The normal and disease proteomic analyses of urinary exosomes are also presented. Thus, this review may provide a valuable reference for future research. PMID:24962155

  17. Developmental changes in the composition of polyadenylated RNA isolated from free and membrane-bound polyribosomes of the rat forebrain, analysed by translation in vitro.

    PubMed Central

    Hall, C; Lim, L

    1981-01-01

    Free and membrane-bound polyribosomes were isolated from the rat forebrain during its development. Polyadenylated RNA [poly(A)+ RNA] was isolated from both fractions, by using oligo(dT)-cellulose chromatography, and its composition studied by translating the poly(A)+ RNA in vitro in reticulocyte lysates. Electrophoretic analysis of the translation products showed that both free and membrane-bound polyribosomal poly(A)+ RNA gave many common components, but that there were also distinct differences in the protein composition of the products of the two fractions. Several proteins, of mol.wts. 39 000, 37 000, 31 000, 27 000 and 17 000, appeared to be products predominantly of free polyribosomal poly(A)+ RNA, whereas others, of mol.wt. 47 000, 33 000, 24 000 and 21 000 were specific to the membrane-bound polyribosomal poly(A)+ RNA fraction. More developmental changes were observed in the translational products of the membrane-bound poly(A)+ RNA fraction. Proteins of mol.wts. 33 000 and 21 000, which were predominant components of the translational products of this fraction when isolated from 10-day and older rats, were not present in translational products derived from preparations isolated from 3-day-old rats. The developmental appearance of these proteins as translational products of the membrane-bound poly(A)+ RNA suggests the appearance of new mRNA species. These transcriptional changes are discussed in relation to processes involved in brain differentiation, including myelination. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:6171267

  18. Simple Method for Constructing RNA Triangle, Square, Pentagon by Tuning Interior RNA 3WJ Angle from 60° to 90° or 108°.

    PubMed

    Khisamutdinov, Emil F; Bui, My Nguyen Hoan; Jasinski, Daniel; Zhao, Zhengyi; Cui, Zheng; Guo, Peixuan

    2015-01-01

    Precise shape control of architectures at the nanometer scale is an intriguing but extremely challenging facet. RNA has recently emerged as a unique material and thermostable building block for use in nanoparticle construction. Here, we describe a simple method from design to synthesis of RNA triangle, square, and pentagon by stretching RNA 3WJ native angle from 60° to 90° and 108°, using the three-way junction (3WJ) of the pRNA from bacteriophage phi29 dsDNA packaging motor. These methods for the construction of elegant polygons can be applied to other RNA building blocks including the utilization and application of RNA 4-way, 5-way, and other multi-way junctions. PMID:25967062

  19. Advances in methods for native expression and purification of RNA for structural studies

    PubMed Central

    Batey, Robert T.

    2014-01-01

    Many RNAs present unique challenges in obtaining material suitable for structural or biophysical characterization. These issues include synthesis of chemically and conformationally homogeneous RNAs, refolding RNA purified using denaturing preparation techniques, and avoiding chemical damage. To address these challenges, new methodologies in RNA expression and purification have been developed seeking to emulate those commonly used for protein purifications. In this review, recent developments in the preparation of high-quality RNA for structural biology and biophysical applications are discussed, with an emphasis on native methods. PMID:24607442

  20. A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans.

    PubMed

    Andachi, Yoshiki; Kohara, Yuji

    2016-07-01

    Whole-mount in situ hybridization (WISH) is an outstanding method to decipher the spatiotemporal expression patterns of microRNAs (miRNAs) and provides important clues for elucidating their functions. The first WISH method for miRNA detection was developed in zebrafish. Although this method was quickly adapted for other vertebrates and fruit flies, WISH analysis has not been successfully used to detect miRNAs in Caenorhabditis elegans Here, we show a novel WISH method for miRNA detection in C. elegans Using this method, mir-1 miRNA was detected in the body-wall muscle where the expression and roles of mir-1 miRNA have been previously elucidated. Application of the method to let-7 family miRNAs, let-7, mir-48, mir-84, and mir-241, revealed their distinct but partially overlapping expression patterns, indicating that miRNAs sharing a short common sequence were distinguishably detected. In pash-1 mutants that were depleted of mature miRNAs, signals of mir-48 miRNA were greatly reduced, suggesting that mature miRNAs were detected by the method. These results demonstrate the validity of WISH to detect mature miRNAs in C. elegans. PMID:27154969

  1. Improved RNA extraction method using the BioMasher and BioMasher power-plus.

    PubMed

    Yamamoto, Takuji; Nakashima, Kentaro; Maruta, Yukio; Kiriyama, Tomomi; Sasaki, Michi; Sugiyama, Shunpei; Suzuki, Kana; Fujisaki, Hitomi; Sasaki, Jun; Kaku-Ushiki, Yuko; Tanida, Masatoshi; Irie, Shinkichi; Hattori, Shunji

    2012-12-01

    The BioMasher is a disposable homogenizer that was developed to homogenize bovine brain tissue for bovine spongiform encephalopathy diagnosis. Capable of preventing the biohazard risk from infectious samples, it also prevents cross-contamination among samples. The BioMasher is thus widely used in biochemical research, especially for RNA extraction. Here, we tested a novel BioMasher application for RNA extraction from animal and plant tissues. We also developed a grinding machine specific for the BioMasher, named the BioMasher Power-Plus. We developed RNA extraction protocols using the BioMasher combined with the BioMasher Power-Plus. We compared RNA extraction efficiency of the BioMasher with that of the FastPrep and the glass homogenizer. Though the RNA extraction efficiency by the BioMasher was nearly equivalent to that of the FastPrep and the glass homogenizer, sample preparation time was shorter for the BioMasher. The utility of RNA extraction by the BioMasher was examined in mouse, rat, and tomato tissue samples. In the rodent tissues, the highest extraction efficiency of total RNA was from liver, with lowest efficiency from fibrous tissues such as muscle. The quality of extracted total RNA was confirmed by agarose gel electrophoresis which produced highly visible clear bands of 18S and 28S rRNAs. Reproducibility among different operators in RNA extraction from tomato roots was improved by using the BioMasher Power-Plus. The BioMasher and BioMasher Power-Plus provide an effective and easy homogenization method for total RNA extraction from some rodent and plant tissues. PMID:22813946

  2. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

    1995-04-11

    A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

  3. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

    1995-01-01

    Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

  4. Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene.

    PubMed

    Coêlho, Mariza M; Ferreira-Nozawa, Monica S; Nozawa, Sérgio R; Santos, André L W

    2011-10-01

    Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla), were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species. PMID:22215973

  5. Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene

    PubMed Central

    Coêlho, Mariza M.; Ferreira-Nozawa, Monica S.; Nozawa, Sérgio R.; Santos, André L.W.

    2011-01-01

    Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla), were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species. PMID:22215973

  6. NSMAP: A method for spliced isoforms identification and quantification from RNA-Seq

    PubMed Central

    2011-01-01

    Background The development of techniques for sequencing the messenger RNA (RNA-Seq) enables it to study the biological mechanisms such as alternative splicing and gene expression regulation more deeply and accurately. Most existing methods employ RNA-Seq to quantify the expression levels of already annotated isoforms from the reference genome. However, the current reference genome is very incomplete due to the complexity of the transcriptome which hiders the comprehensive investigation of transcriptome using RNA-Seq. Novel study on isoform inference and estimation purely from RNA-Seq without annotation information is desirable. Results A Nonnegativity and Sparsity constrained Maximum APosteriori (NSMAP) model has been proposed to estimate the expression levels of isoforms from RNA-Seq data without the annotation information. In contrast to previous methods, NSMAP performs identification of the structures of expressed isoforms and estimation of the expression levels of those expressed isoforms simultaneously, which enables better identification of isoforms. In the simulations parameterized by two real RNA-Seq data sets, more than 77% expressed isoforms are correctly identified and quantified. Then, we apply NSMAP on two RNA-Seq data sets of myelodysplastic syndromes (MDS) samples and one normal sample in order to identify differentially expressed known and novel isoforms in MDS disease. Conclusions NSMAP provides a good strategy to identify and quantify novel isoforms without the knowledge of annotated reference genome which can further realize the potential of RNA-Seq technique in transcriptome analysis. NSMAP package is freely available at https://sites.google.com/site/nsmapforrnaseq. PMID:21575225

  7. Monoclonal antibodies against tyrosyl-tRNA synthetase and its isolated cytokine-like domain.

    PubMed

    Kondratiuk, Iuliia; Khoruzenko, Antonina; Cherednyk, Olga; Filonenko, Valeriy; Kornelyuk, Aleksander

    2013-06-01

    Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH2-terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXC-chemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N- and C-terminal domains of TyrRS. Recombinant TyrRS and its N- and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization. PMID:23750478

  8. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    PubMed

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC). PMID:27045556

  9. Impact of Isolated Hepatitis C Virus (HCV) Core-Specific Antibody Detection and Viral RNA Amplification among HCV-Seronegative Dialysis Patients at Risk for Infection

    PubMed Central

    Barril, Guillermina; Quiroga, Juan A.; Arenas, María Dolores; Espinosa, Mario; García-Fernández, Nuria; Cigarrán, Secundino; Herrero, José A.; del Peso, Gloria; Caro, Pilar; García-Agudo, Rebeca; Amézquita, Yésica; Blanco, Ana; Martínez-Rubio, Pilar; Alcázar, José M.; González-Parra, Emilio; Martín-Gómez, Adoración; Castillo, Inmaculada; Bartolomé, Javier

    2014-01-01

    Amplification of hepatitis C virus (HCV) RNA from blood detected occult HCV infections in 30.9% of 210 HCV-seronegative dialysis patients with abnormal liver enzyme levels that had evaded standard HCV testing practices. Isolated HCV core-specific antibody detection identified three additional anti-HCV screening-negative patients lacking HCV RNA amplification in blood who were considered potentially infectious. Together, these findings may affect management of the dialysis setting. PMID:24850345

  10. Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

    PubMed

    Paparini, Andrea; Gofton, Alexander; Yang, Rongchang; White, Nicole; Bunce, Michael; Ryan, Una M

    2015-01-01

    Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are

  11. SnapShot-Seq: A Method for Extracting Genome-Wide, In Vivo mRNA Dynamics from a Single Total RNA Sample

    PubMed Central

    Boswell, Sarah A.; Cloonan, Nicole; Mullen, Thomas E.; Ling, Joseph J.; Miller, Nimrod; Kuersten, Scott; Ma, Yong-Chao; McCarroll, Steven A.; Grimmond, Sean M.; Springer, Michael

    2014-01-01

    mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment. PMID:24586954

  12. Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR.

    PubMed Central

    Devallois, A; Picardeau, M; Paramasivan, C N; Vincent, V; Rastogi, N

    1997-01-01

    Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare

  13. Polymorphism of the 5' terminal region of Citrus tristeza virus (CTV) RNA: incidence of three sequence types in isolates of different origin and pathogenicity.

    PubMed

    Ayllón, M A; López, C; Navas-Castillo, J; Garnsey, S M; Guerri, J; Flores, R; Moreno, P

    2001-01-01

    Sequences of the 5' terminal region of the genomic RNA from eight isolates of Citrus tristeza virus (CTV) were previously classified into three types (I, II and III), with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. Sequencing of an additional 58 cDNA clones from 15 virus isolates showed that all sequences could be unequivocally assigned to one of the three types previously established. The relative frequency of each sequence type was assessed in 57 CTV isolates of different geographic origin and pathogenic characteristics by RT-PCR with sets of type-specific primers using CTV dsRNA as template. None of the isolates yielded amplification of the type I or II sequences alone, but in 19 of them type III sequences were the only amplification product detected. Within isolates containing more than one sequence type, eight had type II and III sequences, 11 had type I and III sequences, and 19 had sequences of the three types. Isolates containing only type III sequences caused only mild to moderate symptoms in Mexican lime, an indicator species for most CTV isolates, whereas isolates causing stem pitting in sweet orange an/or grapefruit, generally contained sequences type II. None of the sequence types could be traced to a precise geographic area, as all types were detected in isolates from at least nine of the 12 countries from which samples were taken. PMID:11266215

  14. Isolation Method (direct plating or enrichment) does not affect Antimicrobial Susceptibility of Campylobacter from Chicken Carcasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine if Campylobacter isolation method influenced antimicrobial susceptibility results, the minimum inhibitory concentrations (MIC) of nine antimicrobials were compared for 291 pairs of Campylobacter isolates recovered from chicken carcass rinse samples using direct plating and an enrichment...

  15. Comparing bioinformatic gene expression profiling methods: microarray and RNA-Seq.

    PubMed

    Mantione, Kirk J; Kream, Richard M; Kuzelova, Hana; Ptacek, Radek; Raboch, Jiri; Samuel, Joshua M; Stefano, George B

    2014-01-01

    Understanding the control of gene expression is critical for our understanding of the relationship between genotype and phenotype. The need for reliable assessment of transcript abundance in biological samples has driven scientists to develop novel technologies such as DNA microarray and RNA-Seq to meet this demand. This review focuses on comparing the two most useful methods for whole transcriptome gene expression profiling. Microarrays are reliable and more cost effective than RNA-Seq for gene expression profiling in model organisms. RNA-Seq will eventually be used more routinely than microarray, but right now the techniques can be complementary to each other. Microarrays will not become obsolete but might be relegated to only a few uses. RNA-Seq clearly has a bright future in bioinformatic data collection. PMID:25149683

  16. Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples

    NASA Technical Reports Server (NTRS)

    Sundaram, Shivshankar; Prabhakarpandian, Balabhaskar; Pant, Kapil; Wang, Yi

    2014-01-01

    RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads

  17. An RNA secondary structure prediction method based on minimum and suboptimal free energy structures.

    PubMed

    Fu, Haoyue; Yang, Lianping; Zhang, Xiangde

    2015-09-01

    The function of an RNA-molecule is mainly determined by its tertiary structures. And its secondary structure is an important determinant of its tertiary structure. The comparative methods usually give better results than the single-sequence methods. Based on minimum and suboptimal free energy structures, the paper presents a novel method for predicting conserved secondary structure of a group of related RNAs. In the method, the information from the known RNA structures is used as training data in a SVM (Support Vector Machine) classifier. Our method has been tested on the benchmark dataset given by Puton et al. The results show that the average sensitivity of our method is higher than that of other comparative methods such as CentroidAlifold, MXScrana, RNAalifold, and TurboFold. PMID:26100179

  18. Characterizing a novel and sensitive method to measure dsRNA in soil.

    PubMed

    Fischer, Joshua R; Zapata, Fatima; Dubelman, Samuel; Mueller, Geoffrey M; Jensen, Peter D; Levine, Steven L

    2016-10-01

    Performing environmental assessments for double-stranded RNA-based agricultural products require the development of sensitive and selective methods to measure biodegradation rates of dsRNAs. We developed and characterized a novel analytical procedure that uses a molecular hybridization assay (QuantiGene(®)) to accurately measure dsRNA extracted from diverse soils. In this report, we utilize this method to demonstrate that two dsRNAs with distinct size, structure, and sequence degrade rapidly in soil with indistinguishable kinetics. PMID:27441991

  19. Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems.

    PubMed

    Silva, D V; Branco, S M J; Holanda, I S A; Royaert, S; Motamayor, J C; Marelli, J P; Corrêa, R X

    2016-01-01

    Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material. PMID:26985935

  20. A method for obtaining RNA from Hemileia vastatrix appressoria produced in planta, suitable for transcriptomic analyses.

    PubMed

    Loureiro, Andreia; Gil Azinheira, Helena; do Céu Silva, Maria; Talhinhas, Pedro

    2015-11-01

    Appressoria are the first infection structures developed by rust fungi and require specific topographic signals from the host for their differentiation. The ease in obtaining appressoria in vitro for these biotrophic fungi led to studies concerning gene expression and gene discovery at appressorial level, avoiding the need to distinguish plant and fungal transcripts. However, in some pathosystems, it was observed that gene expression in appressoria seems to be influenced by host-derived signals, suggesting that transcriptomic analyses performed from in planta differentiated appressoria would be potentially more informative than those from in vitro differentiated appressoria. Nevertheless analysing appressorial RNA obtained from in planta samples is often hampered by an excessive dilution of fungal RNA within plant RNA, besides uncertainty regarding the fungal or plant origin of RNA from highly conserved genes. To circumvent these difficulties, we have recovered Hemileia vastatrix appressoria from Arabica coffee leaf surface using a film of nitrocellulose dissolved in butyl and ethyl acetates (nail polish), and extracted fungal RNA from the polish peel. RNA thus obtained is of good quality and usable for cDNA synthesis and transcriptomic (quantitative PCR) studies. This method could provide the means to investigate specific host-induced appressoria-related fungal pathogenicity factors. PMID:26466882

  1. Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods.

    PubMed

    Bizarro, C V; Alemany, A; Ritort, F

    2012-08-01

    RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na(+)]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg(2+) salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs. PMID:22492710

  2. Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.

    PubMed

    Lin, Yizhu; May, Gemma E; Joel McManus, C

    2015-01-01

    It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecules are currently unknown (Hennelly & Sanbonmatsu, 2012). While chemical probing can reveal single-stranded regions of RNA, traditional approaches to identify sites of chemical modification are time consuming. Mod-seq is a high-throughput method used to map chemical modification sites on RNAs of any size, including complex mixtures of RNA. In this protocol, we describe preparation of Mod-seq high-throughput sequencing libraries from chemically modified RNA. We also describe a software package "Mod-seeker," which is a compilation of scripts written in Python, for the analysis of Mod-seq data. Mod-seeker returns statistically significant modification sites, which can then be used to aid in secondary structure prediction. PMID:26068740

  3. A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel.

    PubMed

    Liu, Sijun; Bonning, Bryony C; Allen Miller, W

    2006-03-01

    A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors. PMID:16307802

  4. Extraction of RNA from the plant Kalanchoë daigremontiana.

    PubMed

    Garcês, Helena; Sinha, Neelima

    2009-10-01

    This protocol describes how to isolate total RNA from several tissues of Kalanchoë daigremontiana. Total RNA can be isolated by using the TRI-reagent method, scaled up for processing 2 g of tissue, or by using the protocol described here, which gives higher concentrations of high quality RNA. The resulting RNA can be used for various applications including generation of cDNA for reverse transcriptase-PCR (RT-PCR), Northern blots, or other purposes. PMID:20147050

  5. Comparison and optimization of methods for the simultaneous extraction of DNA, RNA, proteins, and metabolites.

    PubMed

    Vorreiter, Fränze; Richter, Silke; Peter, Michel; Baumann, Sven; von Bergen, Martin; Tomm, Janina M

    2016-09-01

    The challenge of performing a time-resolved comprehensive analysis of molecular systems has led to the quest to optimize extraction methods. When the size of a biological sample is limited, there is demand for the simultaneous extraction of molecules representing the four areas of "omics": genomics, transcriptomics, proteomics, and metabolomics. Here we optimized a protocol for the simultaneous extraction of DNA, RNA, proteins, and metabolites and compared it with two existing protocols. Our optimization comprised the addition of a methanol/chloroform metabolite purification before the separation of DNA/RNA and proteins. Extracted DNA, RNA, proteins, and metabolites were quantitatively and/or qualitatively analyzed. Of the three methods, only the newly developed protocol yielded all biomolecule classes of adequate quantity and quality. PMID:27237373

  6. Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105+ hMSCs

    PubMed Central

    Schade, Anna; Müller, Paula; Delyagina, Evgenya; Voronina, Natalia; Skorska, Anna; Lux, Cornelia; Steinhoff, Gustav; David, Robert

    2014-01-01

    Genetic modifications of bone marrow derived human mesenchymal stem cells (hMSCs) using microRNAs (miRs) may be used to improve their therapeutic potential and enable innovative strategies in tissue regeneration. However, most of the studies use cultured hMSCs, although these can lose their stem cell characteristics during expansion. Therefore, we aimed to develop a nonviral miR carrier based on polyethylenimine (PEI) bound to magnetic nanoparticles (MNPs) for efficient miR delivery in freshly isolated hMSCs. MNP based transfection is preferable for genetic modifications in vivo due to improved selectivity, safety of delivery, and reduced side effects. Thus, in this study different miR/PEI and miR/PEI/MNP complex formulations were tested in vitro for uptake efficiency and cytotoxicity with respect to the influence of an external magnetic field. Afterwards, optimized magnetic complexes were selected and compared to commercially available magnetic vectors (Magnetofectamine, CombiMag). We found that all tested transfection reagents had high miR uptake rates (yielded over 60%) and no significant cytotoxic effects. Our work may become crucial for virus-free introduction of therapeutic miRs as well as other nucleic acids in vivo. Moreover, in the field of targeted stem cell therapy nucleic acid delivery prior to transplantation may allowfor initial cell modulation in vitro. PMID:24799915

  7. New miRNA labeling method for bead-based quantification

    PubMed Central

    2010-01-01

    Background microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. Results Here we have applied with an innovative approach, the Luminex® xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. Conclusions We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex® xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies. PMID:20553585

  8. RNA-sequencing data analysis of uterus in ovariectomized rats fed with soy protein isolate,17B-estradiol and casein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This data file describes the bioinformatics analysis of uterine RNA-seq data comparing genome wide effects of feeding soy protein isolate compared to casein to ovariectomized female rats age 64 days relative to treatment of casein fed rats with 5 ug/kg/d estradiol and relative to rats treated with e...

  9. Preparative-scale isolation and purification of procaryotic and eucaryotic ribosomal 5 S RNA: Bacillus subtilis, Neurospora crassa, and wheat germ.

    PubMed

    Li, S J; Chang, L H; Chen, S; Marshall, A G

    1984-05-01

    Ribosomal 5 S RNA from three different organisms has been isolated in high yield and purity. Without prior isolation of ribosomes, a presoak in buffer followed by phenol extraction, DE-32 ion-exchange chromatography, and Sephadex G-75 gel-permeation chromatography yields at least 5-10 mg of electrophoretically homogeneous 5 S RNA from 100 g of cells. Ribonuclease activity is eliminated by various combinations of low temperature, sodium dodecyl sulfate, phenol, and bentonite. High-molecular-weight contaminants are suppressed by either 65 degrees C heat treatment or lowered sodium dodecyl sulfate concentration. For the eucaryotes, 5.8 S RNA contamination is reduced either by low temperature in the initial solubilization or by postponing 65 degrees C heat treatment until after the phenol extraction step. PMID:6204554

  10. A microfluorometric method for quantifying RNA and DNA in terrestrial insects

    PubMed Central

    Kyle, M.; Watts, T.; Schade, J.; Elser, J.J.

    2003-01-01

    Evidence is accumulating for a mechanistic linkage between body phosphorus content and growth and reproduction of individual organisms, due in part to variation in allocation of resources to ribosomal RNA. Testing this connection requires reliable methods of quantifying the nucleic acid content of individual organisms. Although methods for quantifying nucleic acids are available for a wide array of organisms, adaptation of such methods for study of insects has been neglected. Sensitive stains and high throughput fluorometric measurements are now available that substantially improve past methodologies. Here we present methods for the extraction and quantification of insect RNA and DNA based on the use of N-lauroylsarcosine and sonication for extraction, the nucleases RNase and DNase, and the use of microplate fluorescent assays to quantify nucleic acids as percent of body weight in insects. We illustrate the method using Drosophila and curculionid weevils. PMID:15841218

  11. Evaluation of Phenotypic and Genotypic Methods for Subtyping Campylobacter jejuni Isolates from Humans, Poultry, and Cattle

    PubMed Central

    Nielsen, Eva Møller; Engberg, Jørgen; Fussing, Vivian; Petersen, Lise; Brogren, Carl-Henrik; On, Stephen L. W.

    2000-01-01

    Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods. PMID:11015406

  12. A Rapid Method for BrdU Immunostaining in Bovine Mammary Cryosections that Retains RNA Quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid method of 5-bromo-2’-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue, while preserving RNA quality of the stained section. BrdU is a thymidine analog that is incorporated into DNA of proliferating cells and thus serves as a proliferation marker. Im...

  13. A mass spectrometry-based method for direct determination of pseudouridine in RNA.

    PubMed

    Yamauchi, Yoshio; Nobe, Yuko; Izumikawa, Keiichi; Higo, Daisuke; Yamagishi, Yoko; Takahashi, Nobuhiro; Nakayama, Hiroshi; Isobe, Toshiaki; Taoka, Masato

    2016-04-01

    Pseudouridine (5-ribosyluracil, Ψ) is the only 'mass-silent' nucleoside produced by post-transcriptional RNA modification. We describe here a novel mass spectrometry (MS)-based method for direct determination of Ψ in RNA. The method assigns a Ψ-containing nucleolytic RNA fragment by an accurate measurement of a signature doubly dehydrated nucleoside anion ([C9H7N2O4](1-),m/z207.04) produced by collision-induced dissociation MS, and it determines the Ψ-containing nucleotide sequence by pseudo-MS(3), i.e. in-source fragmentation followed by MS(2) By applying this method, we identified all of the known Ψs in the canonical human spliceosomal snRNAs and, unexpectedly, found two previously unknown Ψs in the U5 and U6 snRNAs. Because the method allows direct determination of Ψ in a subpicomole quantity of RNA, it will serve as a useful tool for the structure/function studies of a wide variety of non-coding RNAs. PMID:26673725

  14. Two methods for full-length RNA sequencing for low quantities of cells and single cells

    PubMed Central

    Pan, Xinghua; Durrett, Russell E.; Zhu, Haiying; Tanaka, Yoshiaki; Li, Yumei; Zi, Xiaoyuan; Marjani, Sadie L.; Euskirchen, Ghia; Ma, Chao; LaMotte, Robert H.; Park, In-Hyun; Snyder, Michael P.; Mason, Christopher E.; Weissman, Sherman M.

    2013-01-01

    The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3′ biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling. PMID:23267071

  15. Methods and Devices for Micro-Isolation, Extraction, and/or Analysis of Microscale Components

    NASA Technical Reports Server (NTRS)

    Kartalov, Emil P. (Inventor); Shibata, Darryl (Inventor); Taylor, Clive (Inventor); Wade, Lawrence A. (Inventor)

    2014-01-01

    Provided herein are devices and methods for the micro-isolation of biological cellular material. A micro-isolation apparatus described can comprise a photomask that protects regions of interest against DNA-destroying illumination. The micro-isolation apparatus can further comprise photosensitive material defining access wells following illumination and subsequent developing of the photosensitive material. The micro-isolation apparatus can further comprise a chambered microfluidic device comprising channels providing access to wells defined in photosensitive material. The micro-isolation apparatus can comprise a chambered microfluidic device without access wells defined in photosensitive material where valves control the flow of gases or liquids through the channels of the microfluidic device. Also included are methods for selectively isolating cellular material using the apparatuses described herein, as are methods for biochemical analysis of individual regions of interest of cellular material using the devices described herein. Further included are methods of making masking arrays useful for the methods described herein.

  16. Isolation and expression analysis of four HD-ZIP III family genes targeted by microRNA166 in peach.

    PubMed

    Zhang, C H; Zhang, B B; Ma, R J; Yu, M L; Guo, S L; Guo, L

    2015-01-01

    MicroRNA166 (miR166) is known to have highly conserved targets that encode proteins of the class III homeodomain-leucine zipper (HD-ZIP III) family, in a broad range of plant species. To further understand the relationship between HD-ZIP III genes and miR166, four HD-ZIP III family genes (PpHB14, PpHB15, PpHB8, and PpREV) were isolated from peach (Prunus persica) tissue and characterized. Spatio-temporal expression profiles of the genes were analyzed. Genes of the peach HD-ZIP III family were predicted to encode five conserved domains. Deduced amino acid sequences and tertiary structures of the four peach HD-ZIP III genes were highly conserved, with corresponding genes in Arabidopsis thaliana. The expression level of four targets displayed the opposite trend to that of miR166 throughout fruit development, with the exception of PpHB14 from 35 to 55 days after full bloom (DAFB). This finding indicates that miR166 may negatively regulate its four targets throughout fruit development. As for leaf and phloem, the same trend in expression level was observed between four targets and miR166 from 75 to 105 DAFB. However, the opposite trend was observed for the transcript level between four targets and miR166 from 35 to 55 DAFB. miRNA166 may negatively regulate four targets in some but not all developmental stages for a given tissue. The four genes studied were observed to have, exactly or generally, the same change tendency as individual tissue development, a finding that suggests genes of the HD-ZIP III family in peach may have complementary or cooperative functions in various tissues. PMID:26535732

  17. The genetic diversity of genus Bacillus and the related genera revealed by 16s rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

    PubMed Central

    Cihan, Arzu Coleri; Tekin, Nilgun; Ozcan, Birgul; Cokmus, Cumhur

    2012-01-01

    Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4–100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies. PMID:24031834

  18. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    PubMed Central

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-01-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera. PMID:9097423

  19. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    PubMed

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  20. MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

    PubMed Central

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Gait, Michael J.

    2011-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2′-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs. PMID:21441346

  1. MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection.

    PubMed

    Torres, Adrian G; Fabani, Martin M; Vigorito, Elena; Gait, Michael J

    2011-05-01

    MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs. PMID:21441346

  2. Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae.

    PubMed Central

    Wittekind, M; Dodd, J; Vu, L; Kolb, J M; Buhler, J M; Sentenac, A; Nomura, M

    1988-01-01

    The isolation and characterization of temperature-sensitive mutations in RNA polymerase I from Saccharomyces cerevisiae are described. A plasmid carrying RPA190, the gene encoding the largest subunit of the enzyme, was subjected to in vitro mutagenesis with hydroxylamine. Using a plasmid shuffle screening system, five different plasmids were isolated which conferred a temperature-sensitive phenotype in haploid yeast strains carrying the disrupted chromosomal RPA190 gene. These temperature-sensitive alleles were transferred to the chromosomal RPA190 locus for mapping and physiology experiments. Accumulation of RNA was found to be defective in all mutant strains at the nonpermissive temperature. In addition, analysis of pulse-labeled RNA from two mutant strains at 37 degrees C showed that the transcription of rRNA genes was decreased, while that of 5S RNA was relatively unaffected. RNA polymerase I was partially purified from several of the mutant strains grown at the nonpermissive temperature and was shown to be deficient when assayed in vitro. Fine-structure mapping and sequencing of the mutant alleles demonstrated that all five mutations were unique. The rpa190-1 and rpa190-5 mutations are tightly clustered in region I (S.S. Broyles and B. Moss, Proc. Natl. Acad. Sci. USA 83:3141-3145, 1986), the putative zinc-binding region that is common to all eucaryotic RNA polymerase large subunits. The rpa190-3 mutation is located between regions III and IV, and a strain carrying it behaves as a mutant that is defective in the synthesis of the enzyme. This mutation lies within a previously unidentified segment of highly conserved amino acid sequence homology that is shared among the largest subunits of eucaryotic nuclear RNA polymerases. Another temperature-sensitive mutation, rpa190-2, creates a UGA nonsense codon. Images PMID:3054507

  3. A novel method to scale up fungal endophyte isolations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Estimations of species diversity are influenced by sampling intensity which in turn is influenced by methodology. For fungal endophyte diversity studies, the methodology includes surface-sterilization prior to isolation of endophytes. Surface-sterilization is an essential component of fungal endophy...

  4. High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols.

    PubMed

    Nadiya, Fasiludeen; Anjali, Narayanannair; Gangaprasad, Appukuttannair; Sabu, Kalluvettankuzhy Krishnannair

    2015-09-15

    Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms. PMID:26048648

  5. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons.

    PubMed

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the "in vivo" transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs. PMID:27047347

  6. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons

    PubMed Central

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I.; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the “in vivo” transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs. PMID:27047347

  7. Diversity of endophytic bacteria in Malaysian plants as revealed by 16S rRNA encoding gene sequence based method of bacterial identification☆

    PubMed Central

    Loh, Chye Ying; Tan, Yin Yin; Rohani, Rahim; Weber, Jean-Frédéric F.; Bhore, Subhash Janardhan

    2013-01-01

    Bacterial endophytes do have several potential applications in pharmacy, medicine and agricultural biotech industry. The main objective of this study was to understand types of bacterial endophytes associated with dicotyledonous (dicot) and monocotyledonous (monocot) plant species. Isolation of the endophytic bacteria was performed using surface-sterilized various tissue samples, and identification of the endophytic bacterial isolates (EBIs) was completed using 16S rRNA encoding gene sequence similarity based method. In total, 996 EBIs were isolated and identified from 1055 samples of 31 monocot and 65 dicot plant species from Peninsular Malaysia. The 996 EBIs represented 71 different types of bacterial species. Twelve (12) out of 71 species are reported as endophytes for the first time. We conclude that diverse types of bacterial endophytes are associated with dicot and monocot plants, and could be useful in pharmacy, medicine and agricultural biotechnology for various potential applications. PMID:24396249

  8. RAPD analysis and sequencing of ITS1/5.8S rRNA/ITS2 and Fe-hydrogenase as tools for genetic classification of potentially pathogenic isolates of Trichomonas gallinae.

    PubMed

    Sansano-Maestre, José; Martínez-Herrero, María Del Carmen; Garijo-Toledo, María Magdalena; Gómez-Muñoz, María Teresa

    2016-08-01

    Trichomonas gallinae is a worldwide parasite that causes oropharyngeal avian trichomonosis. During eight years, 60 axenic isolates were obtained from different bird species and characterized by three molecular methods: RAPD analysis and PCR-sequencing of ITS1/5.8S rRNA/ITS2 fragment and Fe-hydrogenase gene. We have found two genotypes of ITS1/5.8S rRNA/ITS2 widely distributed among bird populations, a new variant and also two sequences with mixed pattern. Genotype ITS-OBT-Tg-1 was associated with the presence of gross lesions in birds. We have found eight genotypes of the Fe-hydrogenase (A1, A2, C2, C2.1, C4, C5, C6 and C7), three of them are new reports (C5, C6 and C7), and also three sequences with mixed pattern. Subtype A1 of the Fe-hydrogenase was also related with the presence of lesions. RAPD analyses included most of the strains isolated from animals with lesions in one of the sub-clusters. Potentially pathogenic isolates of T. gallinae obtained in this study fulfill the following criteria with one exception: isolated from lesions+ITS-OBT-Tg-1 genotype+FeHyd A1+RAPD sub-cluster I2. PMID:27473993

  9. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  10. A structure-based flexible search method for motifs in RNA.

    PubMed

    Veksler-Lublinsky, Isana; Ziv-Ukelson, Michal; Barash, Danny; Kedem, Klara

    2007-09-01

    The discovery of non-coding RNA (ncRNA) motifs and their role in regulating gene expression has recently attracted considerable attention. The goal is to discover these motifs in a sequence database. Current RNA motif search methods start from the primary sequence and only then take into account secondary structure considerations. One can think of developing a flexible structure-based motif search method that will filter datasets based on secondary structure first, while allowing extensive primary sequence factors and additional factors such as potential pseudoknots as constraints. Since different motifs vary in structure rigidity and in local sequence constraints, there is a need for algorithms and tools that can be fine-tuned according to the searched RNA motif, but differ in their approach from the RNAMotif descriptor language. We present an RNA motif search tool called STRMS (Structural RNA Motif Search), which takes as input the secondary structure of the query, including local sequence and structure constraints, and a target sequence database. It reports all occurrences of the query in the target, ranked by their similarity to the query, and produces an html file that displays graphical images of the predicted structures for both the query and the candidate hits. Our tool is flexible and takes into account a large number of sequence options and existence of potential pseudoknots as dictated by specific queries. Our approach combines pre-folding and an O(m n) RNA pattern matching algorithm based on subtree homeomorphism for ordered, rooted trees. An O(n(2) log n) extension is described that allows the search engine to take into account the pseudoknots typical to riboswitches. We employed STRMS in search for both new and known RNA motifs (riboswitches and tRNAs) in large target databases. Our results point to a number of additional purine bacterial riboswitch candidates in newly sequenced bacteria, and demonstrate high sensitivity on known riboswitches and t

  11. A comparison of Arcobacter isolation methods and the diversity of Arcobacter spp. in Cheshire, UK.__________________________________________

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: The aims of this study were firstly to compare five published methods for the isolation of Arcobacter spp. from animal faeces in order to determine the most sensitive and specific method. Secondly, we analyzed the resulting isolates by multi-locus sequence typing (MLST) in order to investiga...

  12. A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

    PubMed Central

    Fuchs, Anna-Lisa; Neu, Ancilla; Sprangers, Remco

    2016-01-01

    The eukaryotic mRNA 5′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and high-resolution NMR spectroscopy. In combination with 13C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA. PMID:27368341

  13. A general method for rapid and cost-efficient large-scale production of 5' capped RNA.

    PubMed

    Fuchs, Anna-Lisa; Neu, Ancilla; Sprangers, Remco

    2016-09-01

    The eukaryotic mRNA 5' cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and high-resolution NMR spectroscopy. In combination with (13)C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA. PMID:27368341

  14. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

    PubMed Central

    Witwer, Kenneth W.; Buzás, Edit I.; Bemis, Lynne T.; Bora, Adriana; Lässer, Cecilia; Lötvall, Jan; Nolte-‘t Hoen, Esther N.; Piper, Melissa G.; Sivaraman, Sarada; Skog, Johan; Théry, Clotilde; Wauben, Marca H.; Hochberg, Fred

    2013-01-01

    The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. PMID:24009894

  15. A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.

    PubMed

    Kaelberer, Melanie Maya; Jordt, Sven-Eric

    2016-01-01

    Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined. PMID:27168016

  16. Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates.

    PubMed

    Lee, Min Jung; Jang, Sook Jin; Li, Xue Min; Park, Geon; Kook, Joong-Ki; Kim, Min Jung; Chang, Young-Hyo; Shin, Jong Hee; Kim, Soo Hyun; Kim, Dong-Min; Kang, Seong-Ho; Moon, Dae-Soo

    2014-01-01

    Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and blaOXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter. PMID:24157058

  17. A method of isolating treadmill shock and vibration on spacecraft

    NASA Technical Reports Server (NTRS)

    Thornton, William E.

    1989-01-01

    A major problem is currently felt to exist in the implementation of materials processing on a spacecraft. Crystal growers place requirements of one micro-g or less on the vehicle. Simple math produces startling figures for such a restriction e.g., for each ton of vehicle mass with 10(-6) g acceleration limit; Perturbing Force limit, F = .002 lb. For each 10(5) lbs F = 0.1 lb. For each 10(6) lbs F = 1.0 lb. Forces generated by normal human movement on spacecraft of 5x10(5) pounds weight are on an order-of-magnitude greater than allowed by this specification and forces generated by locomotion on a treadmill are more than two orders-of-magnitude greater. Other exercises and normal onboard functions generate forces in between. To accommodate many essential functions it is obvious that even on a vehicle as large as Space Station, a reduction of more than two orders of magnitude in force is required. Commonly used passive shock and vibration isolation devices are complex, heavy, and also would have difficulty meeting the requirements. However, by a new arrangement, adequate isolation can be obtained. Isolation of the treadmill will be treated since it is considered the most significant disturbance at this time.

  18. A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

    PubMed

    Seifert, Veronica A; Clarke, Benjamin L; Crossland, Janet P; Bemis, Lynne T

    2016-09-01

    A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species. PMID:27349513

  19. Co-ingestion of carbohydrate and whey protein isolates enhance PGC-1α mRNA expression: a randomised, single blind, cross over study

    PubMed Central

    2013-01-01

    Background Whey protein isolates (WPI) supplementation is known to improve resistance training adaptations. However, limited information is available on the effects of WPI plus carbohydrate (CHO) supplementation on endurance training adaptations. Method Six endurance trained male cyclists and triathletes (age 29 ± 4 years, weight 74 ± 2 kg, VO2 max 63 ± 3 ml oxygen. kg-1. Min-1, height 183 ± 5 cm; mean ± SEM) were randomly assigned to one of two dietary interventions in a single blind cross over design; CHO or CHO + WPI. Each dietary intervention was followed for 16 days which included the last 2 days having increased CHO content, representing a CHO loading phase. The dietary interventions were iso-caloric and carbohydrate content matched. On completion of the dietary intervention, participants performed an exercise bout, consisting of cycling for 60 min at 70% VO2 max, followed by time trial to exhaustion at 90% VO2 max and recovered in the laboratory for 6 hours. Blood samples and muscle biopsies were taken at various time points at rest and through the exercise trial and recovery. Results Compared to CHO, CHO + WPI increased plasma insulin during recovery at 180 mins (P < 0.05) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) mRNA expression at the end of 6 hours of recovery (P < 0.05). Muscle glycogen did not differ between the two trials. Conclusion This study showed co-ingestion of CHO + WPI may have beneficial effects on recovery and adaptations to endurance exercise via, increased insulin response and up regulation of PGC-1α mRNA expression. PMID:23402493

  20. Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes.

    PubMed

    Fujii, Katsuhiko; Kuwahara, Anna; Nakamura, Kanako; Yamashita, Yuki

    2011-08-01

    Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases. PMID:21656138

  1. Evaluation of the impact of RNA preservation methods of spiders for de novo transcriptome assembly.

    PubMed

    Kono, Nobuaki; Nakamura, Hiroyuki; Ito, Yusuke; Tomita, Masaru; Arakawa, Kazuharu

    2016-05-01

    With advances in high-throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost-effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high-quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field-collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to -80°C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater-ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150-bp paired-end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field-collected samples of spiders. PMID:26561354

  2. Differential Expression Analysis for RNA-Seq: An Overview of Statistical Methods and Computational Software

    PubMed Central

    Huang, Huei-Chung; Niu, Yi; Qin, Li-Xuan

    2015-01-01

    Deep sequencing has recently emerged as a powerful alternative to microarrays for the high-throughput profiling of gene expression. In order to account for the discrete nature of RNA sequencing data, new statistical methods and computational tools have been developed for the analysis of differential expression to identify genes that are relevant to a disease such as cancer. In this paper, it is thus timely to provide an overview of these analysis methods and tools. For readers with statistical background, we also review the parameter estimation algorithms and hypothesis testing strategies used in these methods. PMID:26688660

  3. Methods for isolation and viability assessment of biological organisms

    SciTech Connect

    Letant, Sonia Edith; Baker, Sarah Elyse; Bond, Tiziana; Chang, Allan Shih-Ping

    2015-02-03

    Isolation of biological or chemical organisms can be accomplished using a surface enhanced Raman scattering (SERS) system. The SERS system can be a single or a stacked plurality of photonic crystal membranes with noble-metal lined through pores for flowing analyte potentially containing the biological or chemical organisms. The through pores can be adapted to trap individual biological or chemical organisms and emit SERS spectra, which can then be detected by a detector and further analyzed for viability of the biological or chemical organism.

  4. A new isolation method for labyrinthulids using a bacterium, Psychrobacter phenylpyruvicus.

    PubMed

    Yokochi, T; Nakahara, T; Higashihara, T; Yamaoka, M; Kurane, R

    2001-01-01

    A new isolation method for labyrinthulids, marine microbes with spindle-shaped vegetative cells and gliding movement, is presented. The method for isolating labyrinthulids has been found to be more difficult and less reproducible than that for thraustochytrids, classified in the same order. So far serum seawater agar fortified with antibiotics has been proposed to be the best for isolation of labyrinthulids. The method presented here involves placing plant samples on an agar medium on which a marine bacterium, Psychrobacter phenylpyruvicus, has been grown. The new method, which utilizes fallen mangrove leaves as source material, was more than twice as effective as isolation agar medium without the bacterium. The increased effectiveness appears to derive partly from the bacterial colonies' delaying extension of fungal mycelium. The bacterium was more effective for the isolation of labyrinthulids than either the bacterium Shewanella sp. or the yeast Rhodotorula rubra. PMID:14961392

  5. Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences.

    PubMed

    Arahal, D R; Dewhirst, F E; Paster, B J; Volcani, B E; Ventosa, A

    1996-10-01

    Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized. PMID:8837434

  6. Isolation of Inositol Hexaphosphate (IHP)-Degrading Bacteria from Arbuscular Mycorrhizal Fungal Hyphal Compartments Using a Modified Baiting Method Involving Alginate Beads Containing IHP

    PubMed Central

    Hara, Shintaro; Saito, Masanori

    2016-01-01

    Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF. PMID:27383681

  7. Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method

    PubMed Central

    Honda, Shozo; Morichika, Keisuke; Kirino, Yohei

    2016-01-01

    RNA digestions catalyzed by many ribonucleases generate RNA fragments containing a 2′,3′-cyclic phosphate (cP) at their 3′-termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named “cP-RNA-seq” that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3′-termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment, hence subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ~6 d, excluding the time required for sequencing and bioinformatics analyses, such downstream assays are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ~3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5′-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes. PMID:26866791

  8. A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

    PubMed

    Greco, Maria; Sáez, Claudio A; Brown, Murray T; Bitonti, Maria Beatrice

    2014-01-01

    The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg(-1) fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method

  9. Systems and methods for bi-directional energy delivery with galvanic isolation

    SciTech Connect

    Kajouke, Lateef A.

    2013-06-18

    Systems and methods are provided for bi-directional energy delivery. A charging system comprises a first bi-directional conversion module, a second bi-directional conversion module, and an isolation module coupled between the first bi-directional conversion module and the second bi-directional conversion module. The isolation module provides galvanic isolation between the first bi-directional conversion module and the second bi-directional conversion module.

  10. Comparison of methods for Identification of Mycobacterium abscessus and M. chelonae isolates.

    PubMed

    Yakrus, M A; Hernandez, S M; Floyd, M M; Sikes, D; Butler, W R; Metchock, B

    2001-11-01

    Mycobacterium abscessus and Mycobacterium chelonae are two closely related species that are often not distinguished by clinical laboratories despite the fact they cause diseases requiring different treatment regimens. Multilocus enzyme electrophoresis, PCR-restriction fragment length polymorphism analysis of the 65-kDa heat shock protein gene, biochemical tests, and high-performance liquid chromatography of mycolic acids were used to identify 75 isolates as either M. abscessus or M. chelonae that were originally submitted for drug susceptibility testing. Only 36 of these isolates were submitted with an identification at the species level. Using the above methods, 46 of the isolates were found to be M. abscessus and 29 were identified as M. chelonae. Eight isolates originally submitted as M. chelonae were identified as M. abscessus, and one isolate submitted as M. abscessus was found to be M. chelonae. The four identification methods were in agreement in identifying 74 of the 75 isolates. In drug susceptibility testing, all isolates of M. abscessus exhibited resistance to tobramycin (MIC of 8 to > or =16 microg/ml), while all isolates of M. chelonae were susceptible to this drug (MIC of < or = 4 microg/ml). The results suggest that once an identification method is selected, clinical laboratories should be able to easily identify isolates of M. abscessus and M. chelonae. PMID:11682537

  11. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  12. Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing

    PubMed Central

    Xiao, Yongli; Sheng, Zong-Mei; Taubenberger, Jeffery K.

    2015-01-01

    The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus at high coverage was determined in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. PMID:26344216

  13. Prevalence of 16S rRNA Methylase Gene rmtB Among Escherichia coli Isolated from Bovine Mastitis in Ningxia, China.

    PubMed

    Yu, Ting; He, Tao; Yao, Hong; Zhang, Jin-Bao; Li, Xiao-Na; Zhang, Rong-Ming; Wang, Gui-Qin

    2015-09-01

    The aim of this study is to understand the prevalence and molecular characterization of 16S rRNA methylase gene, rmtB, among Escherichia coli strains isolated from bovine mastitis in China. A total of 245 E. coli isolates were collected from bovine mastitis in China between 2013 and 2014 and were screened for 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA) by polymerase chain reaction. About 5.3% (13/245) of the isolates carried the rmtB gene; the isolates were highly resistant to amikacin. Thirteen rmtB-positive strains were analyzed for the presence of extended-spectrum β-lactamase genes (bla(TEM), bla(CTX-M), bla(OXA), and bla(SHV)). All the isolates harbored both bla(TEM-1) and bla(CTX-M-15) genes and two of the isolates were also positive for bla(OXA-1). Pulsed-field gel electrophoresis (PFGE) analysis indicated that the nine rmtB-positive strains belonging to ST10 from one farm showed the similar PFGE pattern, indicating a clonal expansion in this farm. S1-PFGE and Southern blotting showed that 12 isolates harbored the rmtB gene in plasmids of two different sizes (≈45 kb [n=10] and ≈48 kb [n=2]), while only 1 strain harbored the rmtB gene in the chromosome. These plasmids were transferable by conjugation studies, and two isolates from two respective farms carried the same size of plasmid, suggesting that the horizontal transmission of plasmids also contributed to the spread of rmtB gene. This is the first report of prevalence of the 16S rRNA methylase gene rmtB among E. coli isolated from bovine mastitis in China, and rmtB-carrying E. coli may pose a threat to the treatment of bovine mastitis. PMID:26203763

  14. A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

    PubMed Central

    Zhang, Rui; Liu, Shengxue; Chiba, Sotaro; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2014-01-01

    Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5′-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1. PMID:25101066

  15. Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Typhimurium Isolates Obtained from Food Animal Sources

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular characterization (e.g. DNA-based typing methods) of Salmonella isolates is frequently employed to compare and distinguish clinical isolates recovered from animals, foodborne disease and nosocomial infections. In this study, we compared the ability of different phenotyping and genotyping m...

  16. Design and Methods of Large-Scale RNA Interference Screens in Drosophila.

    PubMed

    Zhou, Jia; Tong, Chao

    2016-01-01

    Drosophila is an ideal model system for addressing important questions in biology. The use of RNA interference (RNAi) to knockdown gene expression in fly tissues is both very effective and relatively simple. In the past few decades, genome-wide UAS-RNAi transgenic libraries and thousands of Gal4 strains have been generated and have facilitated large-scale in vivo RNAi screening. Here, we discuss methods for the design and performance of a large-scale in vivo RNAi screen in Drosophila. Furthermore, methods for the validation of results and analysis of data will be introduced. PMID:27581292

  17. Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China.

    PubMed

    Xia, Jing; Sun, Jian; Cheng, Ke; Li, Liang; Fang, Liang-Xing; Zou, Meng-Ting; Liao, Xiao-Ping; Liu, Ya-Hong

    2016-05-30

    A total of 963 non-duplicate Escherichia coli strains isolated from food-producing animals between 2002 and 2012 were screened for the presence of the 16S rRNA methyltransferase genes. Among the positive isolates, resistance determinants to extended spectrum β-lactamases, plasmid-mediated quinolone resistance genes as well as floR and fosA/A3/C2 were detected using PCR analysis. These isolates were further subjected to antimicrobial susceptibility testing, molecular typing, PCR-based plasmid replicon typing and plasmid analysis. Of the 963 E. coli isolates, 173 (18.0%), 3 (0.3%) and 2 (0.2%) were rmtB-, armA- and rmtE-positive strains, respectively. All the 16S rRNA methyltransferase gene-positive isolates were multidrug resistant and over 90% of them carried one or more type of resistance gene. IncF (especially IncFII) and non-typeable plasmids played the main role in the dissemination of rmtB, followed by the IncN plasmids. Plasmids that harbored rmtB ranged in size from 20kb to 340kb EcoRI-RFLP testing of the 109 rmtB-positive plasmids from different years and different origins suggested that horizontal (among diverse animals) and vertical transfer of IncF, non-typeable and IncN-type plasmids were responsible for the spread of rmtB gene. In summary, our findings highlight that rmtB was the most prevalent 16S rRNA methyltransferase gene, which present persistent spread in food-producing animals in China and a diverse group of plasmids was responsible for rmtB dissemination. PMID:27139028

  18. Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

    PubMed Central

    González, Sheyla; Ibáñez, Elena

    2010-01-01

    Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536

  19. Antimicrobial susceptibility testing of Australian isolates of Brachyspira hyodysenteriae using a new broth dilution method.

    PubMed

    Karlsson, Märit; Oxberry, Sophy L; Hampson, David J

    2002-01-01

    The antimicrobial susceptibilities of 76 field isolates of Brachyspira hyodysenteriae from different states of Australia were tested in a newly developed broth dilution procedure. The antimicrobial agents used were tiamulin, valnemulin, tylosin, erythromycin, lincomycin and clindamycin. The results from the broth dilution susceptibility testing of 39 of the isolates were compared with results obtained for the same isolates using the agar dilution method. Amongst the isolates tested by broth dilution, 17 were from three farms and had been collected over a number of years. Their pulsed field gel electrophoresis pattern previously had been determined. The broth dilution technique was simple to use, less labor intensive than agar dilution, and gave clear end points. The results obtained using the two methods generally corresponded well, although in a few cases the MIC obtained by broth dilution were lower than those with agar dilution. For the 76 isolates tested by broth dilution, the MIC(90) (mg/l) was: tiamulin, 1; valnemulin, 0.5; tylosin>256; erythromycin>256; lincomycin, 64 and clindamycin, 16. Only minor differences in susceptibility patterns were found amongst isolates from different Australian states. Over all the isolates, and also amongst the isolates obtained from different years on the three farms, there was no trend for the susceptibility of the isolates to alter with time. PMID:11731165

  20. An efficient method for flanking sequence isolation in barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An adapter ligation method was developed to determine native barley (Hordeum vulgare) sequences flanking Ds insertions and barley ESTs. This method is simple and efficient, with the majority of queries returning valid sequence information. This report describes the protocol in detail, quantifies its...

  1. Principal methods for isolation and identification of soil microbial communities.

    PubMed

    Stefanis, Christos; Alexopoulos, Athanasios; Voidarou, Chrissa; Vavias, Stavros; Bezirtzoglou, Eugenia

    2013-01-01

    Soil microbial populations play crucial role in soil properties and influence below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Operations such as the production of antibiotics and enzymic activities from microorganisms of soil constitute objectives of industry in her effort to cope with the increase of population of earth and disturbance of environment and may ameliorate the effects of global climate change. In the past decades, new biochemical and molecular techniques have been developed in our effort to identify and classify soil bacteria. The goal of measuring the soil microbial diversity is difficult because of the limited knowledge about bacteria species and classification through families and orders. Molecular techniques extend our knowledge about microbial diversity and help the taxonomy of species. Measuring and monitoring soil microbial communities can lead us to better understanding of their composition and function in many ecosystem processes. PMID:22791233

  2. Molecular characterisation of Mycoplasma hyorhinis isolated from pigs using pulsed-field gel electrophoresis and 16S rRNA sequencing

    PubMed Central

    Yamaguti, Maurício; Oliveira, Rosângela C; Marques, Lucas M; Buzinhani, Melissa; Buim, Marcos R; Neto, Renata L; Guimarães, Ana Márcia S; Timenetsky, Jorge

    2015-01-01

    Economic loss in pig breeding is common due to respiratory disorders, and Mycoplasma hyopneumoniae and Mycoplasma hyorhinis, namely, are the most common infectious agents. The aim of this study is to recover these mollicutes and detect their genotypic variations by pulsed-field gel electrophoresis (PFGE) and sequencing the 16 s rRNA gene. One hundred and twenty-six swabs from tonsil and nasal mucus of pigs with respiratory disorders were analysed. A total of 78 lungs were sampled, as well as two trachea and two tonsils obtained from animals with respiratory disorder. A total of 59 isolates were obtained: 1 (1.70 per cent) of M hyopneumoniae, 2 (3.40 per cent) of Mycoplasma flocculare and 56 (94.90 per cent) of M hyorhinis. The PFGE for M hyorhinis showed 10 profiles with enzyme AvaI and 9 profiles with XhoI. A low polymorphism of the 16sRNS gene was detected in M hyorhinis isolates compared with the type strain in the GenBank. M hyorhinis isolates of different herds showed a large heterogenicity with enzymes AvaI and XhoI. The sequencing of the 16S rRNA gene allowed for analysing the interspecific and intraspecific variations of isolated mycoplasmas. PMID:26688737

  3. Molecular characterisation of Mycoplasma hyorhinis isolated from pigs using pulsed-field gel electrophoresis and 16S rRNA sequencing.

    PubMed

    Yamaguti, Maurício; Oliveira, Rosângela C; Marques, Lucas M; Buzinhani, Melissa; Buim, Marcos R; Neto, Renata L; Guimarães, Ana Márcia S; Timenetsky, Jorge

    2015-01-01

    Economic loss in pig breeding is common due to respiratory disorders, and Mycoplasma hyopneumoniae and Mycoplasma hyorhinis, namely, are the most common infectious agents. The aim of this study is to recover these mollicutes and detect their genotypic variations by pulsed-field gel electrophoresis (PFGE) and sequencing the 16 s rRNA gene. One hundred and twenty-six swabs from tonsil and nasal mucus of pigs with respiratory disorders were analysed. A total of 78 lungs were sampled, as well as two trachea and two tonsils obtained from animals with respiratory disorder. A total of 59 isolates were obtained: 1 (1.70 per cent) of M hyopneumoniae, 2 (3.40 per cent) of Mycoplasma flocculare and 56 (94.90 per cent) of M hyorhinis. The PFGE for M hyorhinis showed 10 profiles with enzyme AvaI and 9 profiles with XhoI. A low polymorphism of the 16sRNS gene was detected in M hyorhinis isolates compared with the type strain in the GenBank. M hyorhinis isolates of different herds showed a large heterogenicity with enzymes AvaI and XhoI. The sequencing of the 16S rRNA gene allowed for analysing the interspecific and intraspecific variations of isolated mycoplasmas. PMID:26688737

  4. A novel single-stranded RNA virus isolated from the rice-pathogenic fungus Magnaporthe oryzae with similarity to members of the family Tombusviridae.

    PubMed

    Ai, Ye-Ping; Zhong, Jie; Chen, Chuan-Yuan; Zhu, Hong-Jian; Gao, Bi-Da

    2016-03-01

    Here, we report a novel virus isolated from rice blast fungus, Magnaporthe oryzae, an important plant pathogen. This virus has an RNA genome of 3246 nucleotides. Its genome possesses two in-frame open reading frames (ORFs). The smaller ORF1 encodes a protein with significant similarity to a protein encoded by the ssRNA mycovirus Diaporthe ambigua RNA virus 1 (DaRV1). The larger ORF2 encodes a protein with similarity to RNA-dependent RNA polymerases (RdRp) of DaRV1 and other plant viruses of the family Tombusviridae. In silico analysis and comparisons with DaRV1 genome expression suggest that ORF2 is translated via a readthrough mechanism together with ORF1. Based upon results of this study, this virus, for which the provisional name Magnaporthe oryzae virus A (MoVA) is proposed, belongs to a new virus species. Furthermore, MoVA along with DaRV1 belong to a new taxon of mycoviruses that are evolutionarily related to plant viruses belonging to the family Tombusviridae. PMID:26650038

  5. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

    PubMed

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN. PMID:27059238

  6. Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat.

    PubMed

    Ayyaz, Khadija; Zaheer, Ahmad; Rasul, Ghulam; Mirza, Muhammad Sajjad

    2016-01-01

    The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49±1.04mgL(-1)) and phosphate solubilization (105.50±4.93mgL(-1)) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth. PMID:27133558

  7. Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes.

    PubMed

    Potgieter, A C; Steele, A D; van Dijk, A A

    2002-09-01

    Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Phi12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0.8 to 6.8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5' GUU and UAC 3') and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22-30 cycles compared to 30-35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression. PMID:12185276

  8. Rapid isolation of both double-stranded RNA and PCR-suitable DNA from the obligate biotrophic phytopathogenic fungus Uncinula necator using a commercially available reagent.

    PubMed

    Délye, C; Corio-Costet, M F

    1998-10-01

    A method for rapid extraction of both double-stranded RNA (dsRNA) and DNA from an obligate biotrophic phytopathogenic fungus is described. Lyophilised fungal material is incubated in a commercial guanidium thiocyanate reagent. Proteins and cell debris are centrifuged by chloroform precipitation. After precipitation in isopropanol and washing in 75% ethanol, nucleic acids are resuspended in water (10 microl/mg fungal dry weight). DsRNA is directly visualised by agarose gel electrophoresis. DNA contained in 10-fold dilutions of the samples proved to be suitable for PCR-based experiments. PMID:9779614

  9. Microbial Diversity of the Brine-Seawater Interface of the Kebrit Deep, Red Sea, Studied via 16S rRNA Gene Sequences and Cultivation Methods

    PubMed Central

    Eder, Wolfgang; Jahnke, Linda L.; Schmidt, Mark; Huber, Robert

    2001-01-01

    The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Δ7 and Δ11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface. PMID:11425725

  10. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory.

    PubMed

    Tek, Alex; Korostelev, Andrei A; Flores, Samuel Coulbourn

    2016-01-01

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion. PMID:26673695

  11. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory

    PubMed Central

    Tek, Alex; Korostelev, Andrei A.; Flores, Samuel Coulbourn

    2016-01-01

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion. PMID:26673695

  12. Identification of new 18S rRNA strains of Babesia canis isolated from dogs with subclinical babesiosis.

    PubMed

    Łyp, P; Adaszek, Ł; Furmaga, B; Winiarczyk, S

    2015-01-01

    In this study, we used PCR to detect and characterize B. canis from naturally infected dogs in Poland with subclinical babesiosis by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from ten dogs with subclinical babesiosis. A 559-bp fragment of the B. canis 18S rRNA gene was amplified by PCR. Sequencing of the PCR products led to the identification of a new variant of Babesia canis, differing from the previously detected protozoa genotypes (18S rRNA-A and 18S rRNA-B) with nucleotide substitutions in positions 150 and 151 of the tested gene fragment. The results indicate the emergence within the Polish territory of a new, previously unencountered Babesia canis genotype responsible for the development of subclinical babesiosis. PMID:26618590

  13. Method for encapsulating and isolating hazardous cations, medium for encapsulating and isolating hazardous cations

    DOEpatents

    Wasserman, Stephen R.; Anderson, Kenneth B.; Song, Kang; Yuchs, Steven E.; Marshall, Christopher L.

    1998-01-01

    A method for encapsulating hazardous cations is provided comprising supplying a pretreated substrate containing the cations; contacting the substrate with an organo-silane compound to form a coating on the substrate; and allowing the coating to cure. A medium for containing hazardous cations is also provided, comprising a substrate having ion-exchange capacity and a silane-containing coating on the substrate.

  14. RNA Colony Blot Hybridization Method for Enumeration of Culturable Vibrio cholerae and Vibrio mimicus Bacteria▿

    PubMed Central

    Grim, Christopher J.; Zo, Young-Gun; Hasan, Nur A.; Ali, Afsar; Chowdhury, Wasimul B.; Islam, Atiqul; Rashid, Mohammed H.; Alam, Munirul; Morris, J. Glenn; Huq, Anwar; Colwell, Rita R.

    2009-01-01

    A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be

  15. Microbial communities in the saturated groundwater environment I: Methods of isolation and characterization of heterotrophic bacteria.

    PubMed

    Kölbel-Boelke, J; Tienken, B; Nehrkorn, A

    1988-07-01

    In this paper we present a method of isolation and morphological and physiological characterization of groundwater bacteria based on numerical taxonomy and cluster analysis, and using a miniaturized test system (microtiter plates). Bacteria were isolated randomly on P-agar, and each strain was characterized in regard to 155 features. The media for biochemical differentiation are listed as well as methods of morphological discrimination. 246 strains of heterotrophic and oligotrophic bacteria, isolated from five water samples from different depths of the saturated groundwater area, were used for optimizing media and test reactions. PMID:24201530

  16. A New Method for Extraction of Double-Stranded RNA from Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of high molecular weight double-stranded RNA (dsRNA) in plants is associated with the presence of RNA viruses. DsRNA is stable and can be extracted easily from the majority of plant species and provides an excellent tool for characterization of novel viruses that are recalcitrant to pur...

  17. Method for encapsulating and isolating hazardous cations, medium for encapsulating and isolating hazardous cations

    DOEpatents

    Wasserman, S.R.; Anderson, K.B.; Song, K.; Yuchs, S.E.; Marshall, C.L.

    1998-04-28

    A method for encapsulating hazardous cations is provided comprising supplying a pretreated substrate containing the cations; contacting the substrate with an organo-silane compound to form a coating on the substrate; and allowing the coating to cure. A medium for containing hazardous cations is also provided, comprising a substrate having ion-exchange capacity and a silane-containing coating on the substrate. 3 figs.

  18. Sustained lentiviral-mediated overexpression of microRNA124a in the dentate gyrus exacerbates anxiety- and autism-like behaviors associated with neonatal isolation in rats.

    PubMed

    Bahi, Amine

    2016-09-15

    Autism spectrum disorders (ASD) are highly disabling psychiatric disorders. Despite a strong genetic etiology, there are no efficient therapeutic interventions that target the core symptoms of ASD. Emerging evidence suggests that dysfunction of microRNA (miR) machinery may contribute to the underlying molecular mechanisms involved in ASD. Here, we report a stress model demonstrating that neonatal isolation-induced long-lasting hippocampal elevation of miR124a was associated with reduced expression of its target BDNF mRNA. In addition, we investigated the impact of lentiviral-mediated overexpression of miR124a into the dentate gyrus (DG) on social interaction, repetitive- and anxiety-like behaviors in the neonatal isolation (Iso) model of autism. Rats isolated from the dams on PND 1 to PND 11 were assessed for their social interaction, marble burying test (MBT) and repetitive self-grooming behaviors as adults following miR124a overexpression. Also, anxiety-like behavior and locomotion were evaluated in the elevated plus maze (EPM) and open-field (OF) tests. Results show that, consistent with previously published reports, Iso rats displayed decreased social interaction contacts but increased repetitive- and anxiety-like behaviors. Interestingly, across both autism- and anxiety-like behavioral assays, miR124a overexpression in the DG significantly exacerbated repetitive behaviors, social impairments and anxiety with no effect on locomotor activity. Our novel findings attribute neonatal isolation-inducible cognitive impairments to induction of miR124a and consequently suppressed BDNF mRNA, opening venues for intercepting these miR124a-mediated damages. They also highlight the importance of studying microRNAs in the context of ASD and identify miR124a as a novel potential therapeutic target for improving mood disorders. PMID:27211062

  19. [Isolation and characterization of rapidly labelled high molecular RNA from freely suspended callus cells of parsley (Petroselinum sativum)].

    PubMed

    Seitz, U; Richter, G

    1970-12-01

    By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with (32)P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed. PMID:24500301

  20. Methods for Kinetic and Thermodynamic Analysis of Aminoacyl-tRNA Synthetases

    PubMed Central

    Francklyn, Christopher S.; First, Eric A.; Perona, John J.; Hou, Ya-Ming

    2008-01-01

    The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion. PMID:18241792

  1. Method for encapsulating and isolating hazardous cations, medium for encapsulating and isolating hazardous cations

    SciTech Connect

    Wasserman, S.R.; Anderson, K.B.; Song, K.; Yuchs, S.E.; Marshall, C.L.

    1996-12-31

    The problems associated with the disposal of toxic metals in an environmentally acceptable manner continues to plague industry. Such metals as nickel, vanadium, molybdenum, cobalt, iron, and antimony present physiological and ecological challenges that are best addressed through minimization of exposure and dispersion. A method for encapsulating hazardous cations is provided comprising supplying a pretreated substrate containing the cations; contacting the substrate with an organo-silane compound to form a coating on the substrate; and allowing the coating to cure. A medium for containing hazardous cations is also provided, comprising a substrate having ion-exchange capacity and a silane-containing coating on the substrate.

  2. Efficient oil palm total RNA extraction with a total RNA extraction kit.

    PubMed

    Habib, S H; Saud, H M; Kausar, H

    2014-01-01

    Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem. PMID:24781991

  3. A rabbit pulmonary vein myocyte isolation method based on simultaneous heart and pulmonary vein perfusion.

    PubMed

    Gao, Lin-Lin; Zhang, Miao-Miao; Zhang, Liang-Pin; Yang, Shu-Lin; Yao, Ke-Jun; Song, Yuan-Long

    2016-02-25

    Myocytes in the pulmonary veins (PV) play a pivotal role in the development of paroxysmal atrial fibrillation (AF). It is therefore important to understand physiological characteristics of these cells. Studies on these cells are, however, markedly impeded by the fact that single PV myocytes are very difficult to obtain due to lack of effective isolation methods. In this study, we described a novel PV myocyte isolation method. The key aspect of this method is to establish a combination of retrograde heart perfusion (via the aorta) and anterograde PV perfusion (via the pulmonary artery). With this simultaneous perfusion method, a better perfusion of the PV myocytes can be obtained. As results, the output and viability of single myocytes isolated by simultaneous heart and PV perfusion method were increased compared with those in conventional retrograde heart perfusion method. PMID:26915322

  4. Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

    PubMed

    Heidebrecht, F; Schulz, I; Keller, M; Behrens, S-E; Bader, A

    2009-10-01

    The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture. PMID:19539596

  5. Phylogenetic analysis of complete rRNA gene sequence of Nosema philosamiae isolated from the lepidopteran Philosamia cynthia ricini.

    PubMed

    Zhu, Feng; Shen, Zhongyuan; Xu, Xiaofang; Tao, Hengping; Dong, Shinan; Tang, Xudong; Xu, Li

    2010-01-01

    ABSTRACT. The microsporidian Nosema philosamiae is a pathogen that infects the eri-silkworm Philosamia cynthia ricini. The complete sequence of rRNA gene (4,314 bp) was obtained by polymerase chain reaction amplification with specific primers and sequencing. The sequence analysis showed that the organization of the rRNA of N. philosamiae was similar to the pattern of Nosema bombycis. Phylogenetic analysis of rRNA gene sequences revealed that N. philosamiae had a close relationship with other Nosema species, confirming that N. philosamiae is correctly assigned to the genus Nosema. PMID:20384905

  6. Molecular Diversity of Candida albicans Isolated from Immunocompromised Patients, Based on MLST Method

    PubMed Central

    AFSARIAN, Seyed Mohammad Hosein; BADALI, Hamid; SHOKOHI, Tahereh; NAJAFIPOUR, Sohrab

    2015-01-01

    Background: As regards multilocus sequence typing (MLST) method directly analyze the polymorphism within DNA sequences; we performed the first nationwide study on the genotypic relationships of Candida albicans strains obtained from oropharynx and bronchoalveolar lavage (BAL) samples from immunocompromised patients. Methods: Fourteen epidemiologically unrelated clinical strains of C. albicans were obtained from three hospitals in Mazandaran Province, Iran (2006 to 2012) from seven patients with pulmonary infections and the rest with oropharyngeal samples of immunocompromised patients. Seven loci of housekeeping genes were sequenced for all fourteen isolates. Results: MLST was applied to a subset of 14 unrelated isolates. Seventy-one (2.5%) nucleotide sites were found to be variable. Accordingly, 60 different alleles were identified in seven loci among the isolates, among which two new alleles were obtained. Furthermore, 12 independent diploid sequence types (DSTs) including five novel DSTs were identified. The fourteen unrelated isolates were placed in 10 clonal clusters (CC) while two isolates were singletons, by eBURST analysis. Most of the isolates belonged to CC461 of eBURST analysis from the clade 11 and two isolates assigned to CC172 from the clade 15. Conclusion: Pathogen distribution and relatedness for determining the epidemiology of nosocomial infections is highly recommended for pathogen control methods. PMID:26587501

  7. Chemical synthesis of the hexanucleotide d(A-C-C-A-G-C) required to isolate fibroin mRNA on an affinity column.

    PubMed Central

    Cashion, P; Notman, H; Sathe, G; Cadger, T; Porter, K; Jay, E

    1977-01-01

    The synthesis of the hexanucleotide d)A-C-C-A-G-C), complementary to the 2 major triplets of fibroin mRNA, using the phosphotriester methodology is described. The protected dinucleotides ((MeO)2Tr)dbzA.anC, ((MeO)2Tr)danC.bzA and ((meO)2Tr)dacG.anC were synthesized; the latter two were detritylated and joined in stepwize fashion to the 1st to form the protected hexanucleotide ((MeO)2Tr)dbzA.anC.anC.bzA.acG.anC. The latter was deblocked with NH3 and acid to form the hexanucleotide d(A-C-C-A-G-C). In view of the ability of a prototype affinity column, oligo dC-cellulose, to isolate fibroin mRNA, prospects appear excellent for the d(A-C-C-A-G-C)-cellulose affinity column isolation of fibroin mRNA. PMID:909785

  8. Sulfur-inhibited Thermosphaera aggregans sp. nov., a new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences.

    PubMed

    Huber, R; Dyba, D; Huber, H; Burggraf, S; Rachel, R

    1998-01-01

    Recently, a new procedure was developed which allowed for the first time the isolation of a hyperthermophilic archaeum tracked by 165 rRNA analysis from a terrestrial hot solfataric spring ('Obsidian Pool', Yellowstone National Park, WY, USA). This novel isolate is characterized here. Cells are round cocci with a diameter of 0.2-0.8 micron, occurring singly, in pairs, short chains and in grape-like aggregates. The aggregates exhibit a weak bluish-green fluorescence under UV radiation at 420 nm. The new isolate is an anaerobic obligate heterotroph, using preferentially yeast extract for growth. The metabolic products include CO2, H2, acetate and isovalerate. Growth is observed between 65 and 90 degrees C (optimum: 85 degrees C), from pH 5.0 to 7.0 (optimum: 6.5) and up to 0.7% NaCl. The apparent activation energy for growth is about 149 kJ mol-1. Elemental sulfur or hydrogen inhibits growth. The core lipids consist mainly of acyclic and cyclic glycerol diphytanyl tetraethers. The cell envelope contains a cytoplasmic membrane covered by an amorphous layer of unknown composition; there is no evidence for a regularly arrayed surface-layer protein. The G + C content is 46 mol%. On the basis of 165 rRNA sequence comparisons in combination with morphological, physiological and biochemical properties, the isolate represents a new genus within the Desulfurococcaceae, which has been named Thermosphaera. The type species is Thermosphaera aggregans, the type strain is isolate M11TLT (= DSM 11486T). PMID:9542073

  9. Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting

    PubMed Central

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  10. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  11. A novel cost effective and high-throughput isolation and identification method for marine microalgae

    PubMed Central

    2014-01-01

    Background Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures. Results In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms. Conclusions We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures. PMID:25114712

  12. Comparison of isolation and quantification methods to measure humic-like substances (HULIS) in atmospheric particles

    NASA Astrophysics Data System (ADS)

    Fan, Xingjun; Song, Jianzhong; Peng, Ping'an

    2012-12-01

    Humic-like Substances (HULIS) comprise a significant fraction of the water-soluble organic aerosol mass and influence the cloud microphysical properties and climate effects of aerosols in the atmosphere. In this work, the most frequently used HULIS isolation and quantification methods including ENVI-18, HLB, XAD-8 and DEAE were comparatively characterized with two model standards, ten interfering compounds, and five ambient aerosol samples. Quantification of HULIS is performed with a TOC analyzer, complemented by an investigation of the chemical structure of the extracted fractions by UV-Vis spectroscopy. The results show that the four isolation methods were all characterized by high reliability, high reproducibility, and low limit of detection (LOD), indicating that each method can be used to efficiently recover Suwannee River Fulvic Acid (SRFA) and be applied to the quantification of the lower amount of HULIS in atmospheric particles. The analytical results of the UV-Vis spectra of HULIS fractions isolated also indicate that they are all favorable for extraction of compounds of high UV absorbance, high MW, and high aromaticity and that the DEAE protocol is the most significant one. Compared with the DEAE method that favors extraction of highly UV-absorbing and more aromatic compounds, SRFA isolated by the ENVI-18, HLB, and XAD-8 protocols were more representative of the global matrix. Each method has its own advantages and disadvantages and is suitable for a particular application. No single method is ideal for both isolation and quantification of HULIS in atmospheric samples.

  13. Evaluation of Clarithromycin Resistance Among Iranian Helicobacter pylori Isolates by E-Test and Real-Time Polymerase Chain Reaction Methods

    PubMed Central

    Hakemi Vala, Mojdeh; Eyvazi, Shirin; Goudarzi, Hossein; Sarie, Hamid Reza; Gholami, Mehrdad

    2016-01-01

    Background Helicobacter pylori is an important pathogen of human gastric mucosa. Antibiotic resistance, especially resistance to clarithromycin is a major factor for treatment failure of H. pylori infections. The main mechanism of clarithromycin resistance in these bacteria is related to point mutations in three different locations of 23S rRNA gene. Objectives The aims of this study were to evaluate the resistance rate to clarithromycin among local H. pylori isolates by the E-test method and to determine the profile of point mutation in 23S rRNA by real-time polymerase chain reaction (PCR) method. Patients and Methods Eighty biopsy samples were collected from dyspeptic patients by endoscopy during 2011 - 2012. All samples were homogenized immediately and cultured on supplemented brucella blood agar and incubated under microaerophilic conditions. Further biochemical tests and ureC gene PCR was done for H. pylori confirmation. The H. pylori OC1096 strain was used as the control strain, simultaneously. Frequency of clarithromycin resistance was determined by the E-test method based on the clinical and laboratory standard institute (CLSI) standards. Point mutation profile was determined by real-time PCR and further analysis of melting curve, amplicon sequencing was done continuously. Results From 80 biopsy samples, 20 positive H. pylori isolates were detected and confirmed by biochemical tests and PCR method. Overall, 21.7% of the H. pylori isolates, showed clarithromycin resistance phenotype by use of the E-test. Also, the minimal inhibitory concentration of clarithromycin was determined as ≥ 0.5 mg/L by the E-test method. Only point mutation in the location of A2143G with melting temperature of 54.7°C was observed in all resistant isolates. Conclusions This study showed that the frequency of H. pylori clarithromycin resistance in Iran is relatively high. Since clarithromycin is not commonly used in Iran for H. pylori eradication, the high rate of resistance could

  14. A rapid and simple method for the isolation of pure eosinophilic leukocytes from horse blood.

    PubMed

    Jörg, A; Portmann, P; Fellay, G; Dreyer, J L; Meyer, J

    1978-12-15

    An improved and short method is described for the isolation of intact eosinophilic leukocytes from horse blood with high yield (1--1.5 g/20 l). Viability and purity of the preparations were verified by light and electron microscopy and by the trypan blue exclusion test. Isolated eosinophils were 98--100% pure, intact and viable, and they could be shown to phagocytise immune-complexes. PMID:729750

  15. Methods for Determining Elliptic Flow of Isolated Photons and π0's

    NASA Astrophysics Data System (ADS)

    Danley, Tyler; Phenix Collaboration

    2015-04-01

    We present methods for measurements of second order flow coefficients and derivations of reaction plane dependent efficiencies of isolated photons and π0's in Relativistic Heavy Ion Collisions. The method involves isolation cuts similar to those used in direct photon identification where the energy is summed inside an angular cone and cut if greater than a threshold energy. We show that this will result in a reaction plane dependent efficiency. We derive and verify azimuthal single and two particle correlation functions, including this efficiency, up to harmonic second order. We show that the standard v2 extraction method is only sensitive to an effective v2, which includes the sum of true v2 and the v2 of the isolation efficiency, which is generally negative. We will also present the status of applying these methods to PHENIX √{sNN} = 200GeV Au+Au data. for the PHENIX Collaboration.

  16. Comparative study on human and bovine AT-SC isolation methods.

    PubMed

    Reshak, A H; Shahimin, M M; Buang, F

    2013-11-01

    Mammalian adipose tissue derived stem cells (AT-SC) have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 h and 2 h under 37 °C in 5% CO2 incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 h under 37 °C in CO2 incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 h under 37 °C in CO2 incubator. PMID:24080186

  17. From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging.

    PubMed

    Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

    2016-01-01

    In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

  18. From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

    PubMed Central

    Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

    2016-01-01

    In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

  19. A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications.

    PubMed

    Biscans, Annabelle; Rouanet, Sonia; Vasseur, Jean-Jacques; Dupouy, Christelle; Debart, Françoise

    2016-08-01

    An original post-synthetic method on a solid support was developed to introduce various disulfide bond containing groups at the 2'-OH of oligoribonucleotides (RNAs). It is based on a thiol disulfide exchange reaction between several readily accessible alkyldisulfanyl-pyridine derivatives and 2'-O-acetylthiomethyl RNA in the presence of butylamine. By this strategy, diverse 2'-O-alkyldithiomethyl RNAs were obtained. These modifications provided high nuclease resistance to RNA and were easily removed with glutathione treatment, thus featuring a potential use for siRNA prodrugs. PMID:27356960

  20. An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater.

    PubMed

    Kim, Jinyong; Oh, Euna; Banting, Graham S; Braithwaite, Shannon; Chui, Linda; Ashbolt, Nicholas J; Neumann, Norman F; Jeon, Byeonghwa

    2016-01-01

    Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin), to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with C t value using quantitative real-time PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater. PMID:27617011

  1. An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater

    PubMed Central

    Kim, Jinyong; Oh, Euna; Banting, Graham S.; Braithwaite, Shannon; Chui, Linda; Ashbolt, Nicholas J.; Neumann, Norman F.; Jeon, Byeonghwa

    2016-01-01

    Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin), to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with Ct value using quantitative real-time PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater. PMID:27617011

  2. High sensitive method detection of plant RNA viruses by electrochemiluminescence reverse transcription PCR

    NASA Astrophysics Data System (ADS)

    Tang, Ya-bing; Xing, Da; Zhu, De-bin; Zhou, Xiao-ming

    2007-05-01

    It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method, to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection. Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  3. Improved explant method to isolate umbilical cord-derived mesenchymal stem cells and their immunosuppressive properties.

    PubMed

    Mori, Yuka; Ohshimo, Jun; Shimazu, Takahisa; He, Haiping; Takahashi, Atsuko; Yamamoto, Yuki; Tsunoda, Hajime; Tojo, Arinobu; Nagamura-Inoue, Tokiko

    2015-04-01

    The umbilical cord (UC) has become one of the major sources of mesenchymal stem cells (MSCs). The common explant method of isolating UC-derived MSCs (UC-MSCs) involves mincing the UCs into small fragments, which are then attached to a culture dish bottom from which the MSCs migrate. However, the fragments frequently float up from the bottom of the dish, thereby reducing the cell recovery rate. To overcome this problem, we demonstrate an improved explant method for UC-MSC isolation, which involves the use of a stainless steel mesh (Cellamigo(®); Tsubakimoto Chain Co.), to protect the tissue from floating after the minced fragments are aligned at regular intervals in culture dishes. The culture medium was refreshed every 3 days and the adherent cells and tissue fragments were harvested using trypsin. The number of UC-MSCs isolated from 1 g of UC using the explant method with Cellamigo was 2.9 ± 1.4 × 10(6)/g, which was significantly higher than that obtained without Cellamigo (0.66 ± 0.53 × 10(6)/g) (n = 6, p < 0.01) when cells reached 80-90% confluence. In addition, the processing and incubation time required to reach 80-90% confluence was reduced in the improved explant method compared with the conventional method. The UC-MSCs isolated using the improved method were positive for CD105, CD73, CD90, and HLA class I expression and negative for CD45 and HLA class II expression. The isolated UC-MSCs efficiently inhibited the responder T cells induced by allogeneic dendritic cells in a mixed lymphocyte reaction. Conclusively, we demonstrated that the use of Cellamigo improves the explant method for isolating UC-MSCs. PMID:25220032

  4. Full-length M and L RNA sequences of tospovirus isolate 2009-GZT, which causes necrotic ringspot on tomato in China.

    PubMed

    Zheng, Kuanyu; Liu, Hongguang; Yin, Yueyan; Chen, Tsung-Chi; Yeh, Shyi-Dong; Zhang, Zhongkai; Dong, Jiahong

    2016-05-01

    The virus isolate 2009-GZT, collected from tomato in Guizhou province of China, was identified as a new member of the genus Tospovirus based on its S RNA sequence. Because its provisional name, "tomato necrotic spot virus" (TNSV), was identical to an already existing member of the genus Ilarvirus, 2009-GZT was renamed "tomato necrotic spot-associated virus" (TNSaV). In this study, the full-length sequences of the genomic M and L RNAs of TNSaV were determined and analyzed. The M RNA has 4,773 nucleotides (nt), encoding the NSm protein of 309 aa (34.4 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1123 aa (128 kDa) in the viral complementary (vc) strand. The NSm and Gn/Gc of TNSaV share the highest aa sequence identity (86.2 % and 86.9 %, respectively) with those of tomato zonate spot virus. The L RNA contains 8,908 nt and codes for the putative RNA-dependent RNA polymerase (RdRp) of 2885 aa (332 kDa) in the vc strand. The RdRp of TNSaV shares the highest aa sequence identity (85.2 %) with that of calla lily chlorotic spot virus (CCSV). Serological assays showed that TNSaV cross-reacts with rabbit antisera against watermelon silver mottle virus (WSMoV) NP and CCSV NP, indicating that TNSaV is a member of the WSMoV serogroup. PMID:26887969

  5. RNA-sequencing data analysis of uterus in ovariectomized rats fed with soy protein isolate, 17β-estradiol and casein

    PubMed Central

    Ronis, Martin J.; Gomez-Acevedo, Horacio; Blackburn, Michael L.; Cleves, Mario A.; Singhal, Rohit; Badger, Thomas M.

    2016-01-01

    This data file describes the bioinformatics analysis of uterine RNA-seq data comparing genome wide effects of feeding soy protein isolate compared to casein to ovariectomized female rats age 64 days relative to treatment of casein fed rats with 5 μg/kg/d estradiol and relative to rats treated with estradiol and also fed soy protein isolate. Complete raw data files were deposited in the gene Expression Omnibus (GEO) at NCBI (http:/www.ncbi.nlm.nih.gov.geo/) under the GEO accession number GEO: GSE69819. Data presented here incudes a summary of the differential expression analysis with top 30 genes up- and down-regulated by soy protein isolate (SPI), estradiol (E2) and SPI+E2. Additional functional annotation analysis of KEGG pathways is also presented for each treatment, together with networks of interaction between those pathways. Further interpretation and discussion of this data can be found in the article “Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats” Ronis et al. (2016) [1]. PMID:27182546

  6. Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage.

    PubMed

    Benítez-Galeano, María José; Rubio, Leticia; Bertalmío, Ana; Maeso, Diego; Rivas, Fernando; Colina, Rodney

    2015-07-01

    Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program. PMID:26205407

  7. Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage

    PubMed Central

    Benítez-Galeano, María José; Rubio, Leticia; Bertalmío, Ana; Maeso, Diego; Rivas, Fernando; Colina, Rodney

    2015-01-01

    Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program. PMID:26205407

  8. RNA-sequencing data analysis of uterus in ovariectomized rats fed with soy protein isolate, 17β-estradiol and casein.

    PubMed

    Ronis, Martin J; Gomez-Acevedo, Horacio; Blackburn, Michael L; Cleves, Mario A; Singhal, Rohit; Badger, Thomas M

    2016-06-01

    This data file describes the bioinformatics analysis of uterine RNA-seq data comparing genome wide effects of feeding soy protein isolate compared to casein to ovariectomized female rats age 64 days relative to treatment of casein fed rats with 5 μg/kg/d estradiol and relative to rats treated with estradiol and also fed soy protein isolate. Complete raw data files were deposited in the gene Expression Omnibus (GEO) at NCBI (http:/www.ncbi.nlm.nih.gov.geo/) under the GEO accession number GEO: GSE69819. Data presented here incudes a summary of the differential expression analysis with top 30 genes up- and down-regulated by soy protein isolate (SPI), estradiol (E2) and SPI+E2. Additional functional annotation analysis of KEGG pathways is also presented for each treatment, together with networks of interaction between those pathways. Further interpretation and discussion of this data can be found in the article "Uterine responses to feeding soy protein isolate and treatment with 17β-estradiol differ in ovariectomized female rats" Ronis et al. (2016) [1]. PMID:27182546

  9. In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models

    PubMed Central

    Shimkunas, Rafael; Jian, Yuwen; Jaradeh, Mark; Chavez, Karen; Chiamvimonvat, Nipavan; Tardiff, Jil C.; Izu, Leighton T.; Ross, Robert S.; Chen-Izu, Ye

    2016-01-01

    Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation–contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods. PMID:27500929

  10. Influence of DNA isolation method on the investigation of archaeal diversity and abundance in biogas plants.

    PubMed

    Theiss, Juliane; Rother, Michael; Röske, Kerstin

    2016-09-01

    Various methods are available for DNA isolation from environmental samples. Because the chemical and biological composition of samples such as soil, sludge, or plant material is different, the effectiveness of DNA isolation can vary depending on the method applied and thus, have a substantial effect on the results of downstream analysis of the microbial community. Although the process of biogas formation is being intensely investigated, a systematic evaluation of kits for DNA isolation from material of biogas plants is still lacking. Since no DNA isolation kit specifically tailored for DNA isolation from sludge of biogas plants is available, this study compares five commercially available kits regarding their influence on downstream analyses such denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR). The results show that not all kits are equally suited for the DNA isolation from samples of different biogas plants, but highly reproducible DGGE fingerprints as well as qPCR results across the tested samples from biogas reactors using different substrate compositions could be produced using selected kits. PMID:27089887

  11. Remote vacuum or pressure sealing device and method for critical isolated systems

    DOEpatents

    Brock, James David; Keith, Christopher D.

    2012-07-10

    A remote vacuum or pressure sealing apparatus and method for making a radiation tolerant, remotely prepared seal that maintains a vacuum or pressure tight seal throughout a wide temperature range. The remote sealing apparatus includes a fixed threaded sealing surface on an isolated system, a gasket, and an insert consisting of a plug with a protruding sample holder. An insert coupling device, provided for inserting samples within the isolated system, includes a threaded fastener for cooperating with the fixed threaded sealing surface on the isolated system. The insert coupling device includes a locating pin for azimuthal orientation, coupling pins, a tooted coaxial socket wrench, and an insert coupling actuator for actuating the coupling pins. The remote aspect of the sealing apparatus maintains the isolation of the system from the user's environment, safely preserving the user and the system from detrimental effect from each respectively.

  12. An improved method for isolation of epithelial and stromal cells from the human endometrium

    PubMed Central

    MASUDA, Ayako; KATOH, Noriko; NAKABAYASHI, Kazuhiko; KATO, Kiyoko; SONODA, Kenzo; KITADE, Mari; TAKEDA, Satoru; HATA, Kenichiro; TOMIKAWA, Junko

    2016-01-01

    We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from the human endometrium. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease treatment and cell suspension conditions to dissociate single cells from the tissue fragments and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 × 106 of EMECs and 2.8 × 106 EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the molecular mechanisms underlying the dynamic changes of the endometrium during the menstrual cycle. PMID:26853786

  13. A conformation-induced fluorescence method for microRNA detection.

    PubMed

    Aw, Sherry S; Tang, Melissa Xm; Teo, Yin Nah; Cohen, Stephen M

    2016-06-01

    MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3' and 5' ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures. PMID:26951376

  14. A conformation-induced fluorescence method for microRNA detection

    PubMed Central

    Aw, Sherry S.; Tang, Melissa XM; Teo, Yin Nah; Cohen, Stephen M.

    2016-01-01

    MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3′ and 5′ ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures. PMID:26951376

  15. Methods for the Study of Long Noncoding RNA in Cancer Cell Signaling

    PubMed Central

    Feng, Yi; Hu, Xiaowen; Zhang, Youyou; Zhang, Dongmei; Li, Chunsheng; Zhang, Lin

    2014-01-01

    With the advances in sequencing technology and transcriptome analysis, it is estimated that up to 75 % of the human genome is transcribed into RNAs. This finding prompted intensive investigations on the biological functions of noncoding RNAs and led to very exciting discoveries of microRNAs as important players in disease pathogenesis and therapeutic applications. Research on long noncoding RNAs (lncRNAs) is in its infancy, yet a broad spectrum of biological regulations has been attributed to lncRNAs. Here, we provide a collection of detailed experimental protocols for lncRNA studies, including lncRNA immunoprecipitation, lncRNA pull-down, lncRNA northern blot analysis, lncRNA in situ hybridization, and lncRNA knockdown. We hope that the information included in this chapter can speed up research on lncRNAs biology and eventually lead to the development of clinical applications with lncRNA as novel prognostic markers and therapeutic targets. PMID:24839023

  16. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

    PubMed Central

    Tenover, F C; Arbeit, R; Archer, G; Biddle, J; Byrne, S; Goering, R; Hancock, G; Hébert, G A; Hill, B; Hollis, R

    1994-01-01

    Fifty-nine Staphylococcus aureus isolates and 1 isolate of Staphylococcus intermedius were typed by investigators at eight institutions by using either antibiograms, bacteriophage typing, biotyping, immunoblotting, insertion sequence typing with IS257/431, multilocus enzyme electrophoresis, restriction analysis of plasmid DNA, pulsed-field or field inversion gel electrophoresis, restriction analysis of PCR-amplified coagulase gene sequences, restriction fragment length polymorphism typing by using four staphylococcal genes as probes, or ribotyping. Isolates from four well-characterized outbreaks (n = 29) and a collection of organisms from two nursing homes were mixed with epidemiologically unrelated stock strains from the Centers for Disease Control and Prevention. Several isolates were included multiple times either within or between the sets of isolates to analyze the reproducibilities of the typing systems. Overall, the DNA-based techniques and immunoblotting were most effective in grouping outbreak-related strains, recognizing 27 to 29 of the 29 outbreak-related strains; however, they also tended to include 3 to 8 epidemiologically unrelated isolates in the same strain type. Restriction fragment length polymorphism methods with mec gene-associated loci were less useful than other techniques for typing oxacillin-susceptible isolates. Phage typing, plasmid DNA restriction analysis, and antibiogram analysis, the techniques most readily available to clinical laboratories, identified 23 to 26 of 29 outbreak-related isolates and assigned 0 to 6 unrelated isolates to outbreak strain types. No single technique was clearly superior to the others; however, biotyping, because it produced so many subtypes, did not effectively group outbreak-related strains of S. aureus. PMID:7908673

  17. Evaluation of RNA quality in fixed and unembedded mouse embryos by different methods.

    PubMed

    Mu, Yuan; Zhou, Hong; Li, Wenyan; Hu, Lichao; Zhang, Yiting

    2013-10-01

    Many miRNAs are highly expressed in spatiotemporal and precise tissue-specific patterns in development. Thus it is necessary to examine their expression pattern in mouse embryos. However, embryos from one pregnant mouse are more than enough for expression analysis such as RT-qPCR, which results in reluctant disposal of remaining embryos. Due to the limitation of short sampling time, it is vitally important to quickly preserve samples to ensure the RNA quality. Thus, it is necessary to develop appropriate methods to fix samples in advance. In this study, two fixatives [methanol/DMSO (4:1) and paraformaldehyde] were applied for embryo (12.5 dpc) fixation and two preservatives (methanol and 30% sucrose) were used for fixed embryo preservation. After storage for one month, the skin, skeletal muscle and brain tissues were dissected from the fixed and unembedded embryos. Total RNAs were extracted by TRIzol® reagent and measured by a spectrophotometer, then were subjected to amplify Actb, Hprt, Gapdh, Rnu6, Snord68 and miR-206-3p by RT-qPCR. Embryos fixed in methanol/DMSO and preserved in 100% methanol at -20°C were able to yield at least 349 bp amplifiable RNA. Although paraformaldehyde fixation and 30% sucrose preservation method only yielded amplicons less than 156 bp, it showed a remarkable ability in preserving small RNAs. Snord68 was expressed stably across skin, skeletal muscle and brain tissues like Rnu6, making its possibility as an internal control for qPCR data normalization. Using Snord68 and/or Rnu6 as internal control, we found that the miR-206-3p expression level in skin was about one quarter of its highest level in the skeletal muscle. Therefore, the techniques in this study would be useful for us to reasonably utilize and preserve precious samples. PMID:23895953

  18. Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA.

    PubMed

    Kedersha, N L; Rome, L H

    1986-09-01

    Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore considerably larger than ribosomes. When subjected to SDS PAGE, these structures, which we refer to as vaults, appear to contain several minor and five major species: Mr 210,000, 192,000, 104,000, 54,000, and 37,000. One of these (Mr 104,000) greatly predominates, accounting for greater than 70% of the total Coomassie Brilliant Blue-staining protein. Another major species of Mr 37,000 has been identified as a species of small RNA of unusual base composition (adenosine 12.0%, guanosine 29.7%, uridine 30.9%, and 27.4% cytidine), which migrates as a single species in urea PAGE between the 5S and 5.8S ribosomal standards, containing approximately 140 bases. Although the RNA constitutes only 4.6% of the entire structure, the large size of the particle requires that each one contains approximately 9 molecules of this RNA. Antibodies prepared against the entire particle are largely specific for the major (Mr 104,000) polypeptide species. Although they do not directly react with the RNA constituent on Western blots, these antibodies immunoprecipitate a 32P-labeled RNA of identical size from metabolically-labeled rat hepatoma cells. Vaults are observed in partially purified fractions from human fibroblasts, murine 3T3 cells, glial cells, and rabbit alveolar macrophages. It therefore appears that these novel ribonucleoprotein structures are broadly distributed among different cell types. The function of vaults is at present unknown. PMID:2943744

  19. Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes.

    PubMed

    Huang, Jianzhong; Aki, Tsunehiro; Yokochi, Toshihiro; Nakahara, Toro; Honda, Daiske; Kawamoto, Seiji; Shigeta, Seiko; Ono, Kazuhisa; Suzuki, Osamu

    2003-01-01

    Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids. PMID:14730428

  20. Evaluation of Methods for Identification and Determination of the Taxonomic Status of Strains Belonging to the Streptococcus porcinus-Streptococcus pseudoporcinus Complex Isolated from Animal, Human, and Dairy Sources

    PubMed Central

    Steigerwalt, Arnold G.; Whitney, Anne M.; Morey, Roger E.; Graziano, James C.; Facklam, Richard R.; Musser, Kimberlee A.; Merquior, Vânia L. C.; Teixeira, Lucia M.

    2012-01-01

    Ninety-seven animal, human, and dairy Streptococcus porcinus or Streptococcus pseudoporcinus isolates in the CDC Streptococcus strain collection were evaluated on the basis of DNA-DNA reassociation, 16S rRNA and rpoB gene sequencing, conventional biochemical and Rapid ID 32 Strep identification methods, and antimicrobial susceptibility testing to determine their taxonomic status, characteristics for species differentiation, antimicrobial susceptibility, and relevance of clinical source. Nineteen of the 97 isolates (1 human, 18 swine) were identified as S. porcinus. The remaining 72 human isolates and 6 dairy isolates were identified as S. pseudoporcinus. The use of 16S rRNA or rpoB gene sequencing was required to differentiate S. porcinus from S. pseudoporcinus. The human and dairy S. pseudoporcinus isolates were biochemically distinct from each other as well as distinct by 16S rRNA and rpoB gene sequencing. Therefore, we propose the subspecies denominations S. pseudoporcinus subsp. hominis subsp. nov. for the human isolates and S. pseudoporcinus subsp. lactis subsp. nov. for the dairy isolates. Most strains were susceptible to the antimicrobials tested, with the exception of tetracycline. Two strains of each species were also resistant to clindamycin and erythromycin and carried the erm(A) (S. pseudoporcinus) or the erm(B) (S. porcinus) gene. S. porcinus was identified from a single human isolate recovered from a wound in an abattoir worker. S. pseudoporcinus was primarily isolated from the genitourinary tract of women but was also associated with blood, placental, and wound infections. Isolates reacting with group B antiserum and demonstrating wide beta-hemolysis should be suspected of being S. pseudoporcinus and not S. agalactiae. PMID:22933599

  1. Vacuum-isolation vessel and method for measurement of thermal noise in microphones

    NASA Technical Reports Server (NTRS)

    Zuckerwar, Allan J. (Inventor); Ngo, Kim Chi T. (Inventor)

    1992-01-01

    The vacuum isolation vessel and method in accordance with the present invention are used to accurately measure thermal noise in microphones. The apparatus and method could be used in a microphone calibration facility or any facility used for testing microphones. Thermal noise is measured to determine the minimum detectable sound pressure by the microphone. Conventional isolation apparatus and methods have been unable to provide an acoustically quiet and substantially vibration free environment for accurately measuring thermal noise. In the present invention, an isolation vessel assembly comprises a vacuum sealed outer vessel, a vacuum sealed inner vessel, and an interior suspension assembly coupled between the outer and inner vessels for suspending the inner vessel within the outer vessel. A noise measurement system records thermal noise data from the isolation vessel assembly. A vacuum system creates a vacuum between an internal surface of the outer vessel and an external surface of the inner vessel. The present invention thus provides an acoustically quiet environment due to the vacuum created between the inner and outer vessels and a substantially vibration free environment due to the suspension assembly suspending the inner vessel within the outer vessel. The thermal noise in the microphone, effectively isolated according to the invention, can be accurately measured.

  2. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    PubMed

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  3. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

    PubMed Central

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M.

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  4. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    PubMed

    Qiu, Huiling; Zhong, Jiasheng; Luo, Lan; Liu, Nian; Kang, Kang; Qu, Junle; Peng, Wenda; Gou, Deming

    2015-01-01

    DNA vector-encoded Tough Decoy (TuD) miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer), which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD) vector in which only two sets of shorter oligonucleotides (< 60 mer) were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS) of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324) were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo. PMID:26624995

  5. Isolation and Characterization of a Novel Single-Stranded RNA Virus Infectious to a Marine Fungoid Protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)

    PubMed Central

    Takao, Yoshitake; Nagasaki, Keizo; Mise, Kazuyuki; Okuno, Tetsuro; Honda, Daiske

    2005-01-01

    Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decomposers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experiments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 × 103 to 6.4 × 104 infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms. PMID:16085844

  6. Testing promoter activity in the trypanosome genome: isolation of a metacyclic-type VSG promoter, and unexpected insights into RNA polymerase II transcription.

    PubMed

    McAndrew, M; Graham, S; Hartmann, C; Clayton, C

    1998-09-01

    In trypanosomes, most genes are arranged in polycistronic transcription units. Individual mRNAs are generated by 5'-trans splicing and 3' polyadenylation. Remarkably, no regulation of RNA polymerase II transcription has been detected although many RNAs are differentially expressed during kinetoplastid life cycles. Demonstration of specific class II promoters is complicated by the difficulty in distinguishing between genuine promoter activity and stimulation of trans splicing. Using vectors that were designed to allow the detection of low promoter activities in a transcriptionally silent chromosomal context, we isolated a novel trypanosome RNA polymerase I promoter. We were however unable to detect class II promoter activity in any tested DNA fragment. We also integrated genes which were preceded by a T3 promoter into the genome of cells expressing bacteriophage T3 polymerase: surprisingly, transcription was alpha-amanitin sensitive. One possible interpretation of these results is that in trypanosomes, RNA polymerase II initiation is favored by genomic accessibility and double-strand melting. PMID:9709032

  7. Technical note: A rapid method for 5-bromo-2'-deoxyuridine (BrdU) immunostaining in bovine mammary cryosections that retains RNA quality.

    PubMed

    Choudhary, R K; Daniels, K M; Evock-Clover, C M; Garrett, W; Capuco, A V

    2010-06-01

    A rapid method of 5-bromo-2'-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue while preserving RNA quality of the stained section. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] fixation (2 min) followed by staining with methyl green (0.5% aqueous; 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline; 4 min incubation), antibody incubation in the presence of RNase inhibitors (5 min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor alpha and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor alpha- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional

  8. A combined dynamic analysis method for geometrically nonlinear vibration isolators with elastic rings

    NASA Astrophysics Data System (ADS)

    Hu, Zhan; Zheng, Gangtie

    2016-08-01

    A combined analysis method is developed in the present paper for studying the dynamic properties of a type of geometrically nonlinear vibration isolator, which is composed of push-pull configuration rings. This method combines the geometrically nonlinear theory of curved beams and the Harmonic Balance Method to overcome the difficulty in calculating the vibration and vibration transmissibility under large deformations of the ring structure. Using the proposed method, nonlinear dynamic behaviors of this isolator, such as the lock situation due to the coulomb damping and the usual jump resulting from the nonlinear stiffness, can be investigated. Numerical solutions based on the primary harmonic balance are first verified by direct integration results. Then, the whole procedure of this combined analysis method is demonstrated and validated by slowly sinusoidal sweeping experiments with different amplitudes of the base excitation. Both numerical and experimental results indicate that this type of isolator behaves as a hardening spring with increasing amplitude of the base excitation, which makes it suitable for isolating both steady-state vibrations and transient shocks.

  9. Co-precipitation of protein and polyester as a method to isolate high molecular weight DNA.

    PubMed

    Dixson, Jamie D

    2005-02-01

    DNA isolation is often the limiting step in genetic analysis using PCR and automated fragment analysis due to low quality or purity of DNA, the need to determine and adjust DNA concentrations after isolation etc. Several protocols have been developed which are either safe and provide good quality DNA or hazardous and provide excellent quality DNA. In this brief communication I describe a new and rapid method of DNA isolation which employs the co-precipitation of protein and polyester, in the presence of acetone, to remove contaminating proteins from a lysed-tissue sample, thus leaving high quality pure DNA. The advantages of this method are increased safety over the phenol:chloroform and the chaotrophic salt methods and increased purity over the salting-out method. Since the concentrations of DNA isolated using this method are relatively consistent regardless of the amount of starting tissue (within limits), adjustments of the DNA concentrations before use as templates in PCR's are not necessary. PMID:16970607

  10. Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551.

    PubMed

    Maneeruttanarungroj, Cherdsak; Incharoensakdi, Aran

    2016-06-01

    Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30-105 s at 70 °C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains. PMID:27116965

  11. Evaluation of Normalization Methods for RNA-Seq Gene Expression Estimation

    PubMed Central

    Wu, Po-Yen; Phan, John H.; Zhou, Fengfeng; Wang, May D.

    2016-01-01

    Statistical inferences on RNA-Seq data, e.g., detecting differential gene expression, are meaningful only after proper normalization. However, there is no consensus for choosing a normalization procedure from among the many existing procedures. We evaluated several RNA-Seq normalization procedures by (1) correlating estimated RNA-Seq expression values to those of microarrays, (2) examining the concordance of stable and differential gene detection between the platforms, and (3) applying the procedures to simulated RNA-Seq data. Results suggested that RNA-Seq normalization procedures have little effect on both inter-platform gene expression correlation as well as inter-platform concordance of genes detected as stably or differentially expressed. However, the results of simulated analysis suggested that some normalization procedures are more robust to changes in distribution of differentially expressed genes. These results may provide guidance for selecting RNA-Seq normalization procedures.

  12. Faster algorithms for RNA-folding using the Four-Russians method

    PubMed Central

    2014-01-01

    Background The secondary structure that maximizes the number of non-crossing matchings between complimentary bases of an RNA sequence of length n can be computed in O(n3) time using Nussinov’s dynamic programming algorithm. The Four-Russians method is a technique that reduces the running time for certain dynamic programming algorithms by a multiplicative factor after a preprocessing step where solutions to all smaller subproblems of a fixed size are exhaustively enumerated and solved. Frid and Gusfield designed an O(n3logn) algorithm for RNA folding using the Four-Russians technique. In their algorithm the preprocessing is interleaved with the algorithm computation. Theoretical results We simplify the algorithm and the analysis by doing the preprocessing once prior to the algorithm computation. We call this the two-vector method. We also show variants where instead of exhaustive preprocessing, we only solve the subproblems encountered in the main algorithm once and memoize the results. We give a simple proof of correctness and explore the practical advantages over the earlier method. The Nussinov algorithm admits an O(n2) time parallel algorithm. We show a parallel algorithm using the two-vector idea that improves the time bound to O(n2logn). Practical results We have implemented the parallel algorithm on graphics processing units using the CUDA platform. We discuss the organization of the data structures to exploit coalesced memory access for fast running times. The ideas to organize the data structures also help in improving the running time of the serial algorithms. For sequences of length up to 6000 bases the parallel algorithm takes only about 2.5 seconds and the two-vector serial method takes about 57 seconds on a desktop and 15 seconds on a server. Among the serial algorithms, the two-vector and memoized versions are faster than the Frid-Gusfield algorithm by a factor of 3, and are faster than Nussinov by up to a factor of 20. The source-code for the

  13. Novel Campylobacter isolation method using hydrophobic grid membrane filter and semisolid medium.

    PubMed

    Valdivieso-Garcia, Alfonso; Harris, Kathleen; Riche, Edward; Campbell, Stephanie; Jarvie, Anne; Popa, Maria; Deckert, Anne; Reid-Smith, Richard; Rahn, Kris

    2007-02-01

    Culture procedures for isolation of thermophilic campylobacters from food matrices are complex, labor intensive, and time-consuming. Most available methods include the use of antibiotics as selective agents to prevent the growth of competing microflora. A simple procedure for isolation of thermophilic campylobacters after enrichment in Rosef's enrichment broth was developed using a hydrophobic grid membrane filter (HGMF) on semisolid medium (SSM). SSM contains no antibiotics, and the HGMF physically separates Campylobacter from the enrichment broth, allowing isolation based on differential motility. The HGMF-SSM method was compared to the Agriculture and Agri-Food Canada Food Safety Procedures Manual (FSPM-10) method (Isolation of Thermophilic Campylobacters from Fresh Pork, Beef Veal, Poultry and Ready-to-Eat Meat Products), which includes the use of selective antibiotics. During the initial study, after enrichment the HGMF-SSM method yielded pure cultures of campylobacters after 16 to 18 h (overnight) compared with 48 h for the FSPM-10 method. Ninety-four turkey samples collected at local retail stores and 38 frozen pig fecal samples were processed by both methods. Thirty-five samples (26.5%) were positive by the HGMF-SSM method; 24 (18.2%) of these positive samples contained Campylobacter jejuni and 11 (8.3%) contained Campylobacter coli. With the FSPM-10 method, 25 samples (18.9%) were positive: 21 (15.9%) with C. jejuni and 4 (3%) with C. coli. For a subsequent field study, only the HGMF-SSM method was used to isolate Campylobacter from 1,200 chicken samples and 454 turkey samples sold at retail. Analysis of five subisolates from various samples indicated that only one type of Campylobacter was recovered by the HGMF-SSM method, as ascertained by MICs for 10 antimicrobials, sequencing of the short variable region of the flaA gene, and fingerprinting based on amplified fragment length polymorphism. The absence of antibiotics in the SSM may explain the higher

  14. Comprehensive analysis of the whole transcriptomes from two different pig breeds using RNA-Seq method.

    PubMed

    Ropka-Molik, Katarzyna; Zukowski, Kacper; Eckert, Robert; Gurgul, Artur; Piórkowska, Katarzyna; Oczkowicz, Maria

    2014-10-01

    Next-generation sequencing RNA-Seq technology is a powerful tool that creates new possibilities for whole-transcriptome analysis. In our study, the RNA-Seq method was applied to analyze global changes in transcriptome from muscle tissue (m. semimembranosus) in two pig breeds (Pietrain and Polish Landrace, PL). The breeds differ in terms of muscularity, growth rate and reproduction traits. Using three different approaches (deseq, cufflinks and edger) and taking into account the most restrictive criteria, 35 genes differentially expressed between Pietrain and PL pigs were identified. In both breeds, the most abundant were transcripts encoding ribosomal and cytoskeletal proteins (TPM3, TCAP, TMOD4, TPM2, TNNC1) and calcium-binding proteins involved in muscle contraction, calcium-mediated signaling or cation transport (CASQ1, MLC2V, SLC25A4, MYL3). In PL pigs, we identified up-regulation of several genes that play crucial roles in reproduction: female gamete generation (BDP1, PTPN21, USP9X), fertilization (EGFR) and embryonic development (CPEB4). In the Pietrain breed, only seven genes were over-expressed (CISH, SPP1, TUBA8, ATP6V1C2, IGKC, predicted LOC100510960 and LOC100626400), and they play important roles in, for example, negative regulation of apoptosis, immune response, cell-cell signaling, cell growth and migration as well as the metabolic process. The functions of the majority of selected genes were consistent with phenotypic variation in investigated breeds; thus, we proposed a new panel of candidate genes that can be associated with economically important pig traits. PMID:24961663

  15. Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon).

    PubMed

    Unajak, Sasimanas; Boonsaeng, Vichai; Jitrapakdee, Sarawut

    2006-10-01

    We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection. PMID:16938476

  16. A method of transmissibility design for dual-chamber pneumatic vibration isolator

    NASA Astrophysics Data System (ADS)

    Lee, Jeung-Hoon; Kim, Kwang-Joon

    2009-06-01

    Dual-chamber pneumatic vibration isolators have a wide range of applications for vibration isolation of vibration-sensitive equipment. Recent advances in precision machine tools and instruments such as medical devices and those related to nano-technology require better isolation performance, which can be efficiently achieved by precise modeling- and design- of the isolation system. This paper discusses an efficient transmissibility design method of a pneumatic vibration isolator wherein a complex stiffness model of a dual-chamber pneumatic spring developed in our previous study is employed. Three design parameters, the volume ratio between the two pneumatic chambers, the geometry of the capillary tube connecting the two pneumatic chambers, and, finally, the stiffness of the diaphragm employed for prevention of air leakage, were found to be important factors in transmissibility design. Based on a design technique that maximizes damping of the dual-chamber pneumatic spring, trade-offs among the resonance frequency of transmissibility, peak transmissibility, and transmissibility in high frequency range were found, which were not ever stated in previous researches. Furthermore, this paper discusses the negative role of the diaphragm in transmissibility design. The design method proposed in this paper is illustrated through experimental measurements.

  17. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

  18. Coumarins in horse chestnut flowers: isolation and quantification by UPLC method.

    PubMed

    Dudek-Makuch, Marlena; Matławska, Irena

    2013-01-01

    The coumarins: scopoletin, esculetin and fraxetin were isolated from the flowers of horse chestnut (Aesculus hippocastanum L., Hippocastanaceae) and identified by spectrophotometric methods (UV, 1H, 13C NMR, ESI-MS). Their content, determined using the Ultra Performance Liquid Chromatography (UPLC), was 0.41, 0.13 and 0.05%, respectively. PMID:23757942

  19. Technical note: A method for isolating glycogen granules from ruminal protozoa for further characterization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Evaluation of physical, compositional, and digestion characteristics of protozoal glycogen is best performed on a pure substrate in order to avoid interference from other cell components. A method for isolating protozoal glycogen without use of detergents was developed. Rumen inoculum was incubated ...

  20. Simple and efficient methods for enrichment and isolation of endonuclease modified cells.

    PubMed

    Moriarity, Branden S; Rahrmann, Eric P; Beckmann, Dominic A; Conboy, Caitlin B; Watson, Adrienne L; Carlson, Daniel F; Olson, Erik R; Hyland, Kendra A; Fahrenkrug, Scott C; McIvor, R Scott; Largaespada, David A

    2014-01-01

    The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner. PMID:24798371

  1. Protein mediated miRNA detection and siRNA enrichment using p19.

    PubMed

    Jin, Jingmin; Cid, Melissa; Poole, Catherine B; McReynolds, Larry A

    2010-06-01

    p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs. PMID:20569217

  2. AuPairWise: A Method to Estimate RNA-Seq Replicability through Co-expression.

    PubMed

    Ballouz, Sara; Gillis, Jesse

    2016-04-01

    In addition to detecting novel transcripts and higher dynamic range, a principal claim for RNA-sequencing has been greater replicability, typically measured in sample-sample correlations of gene expression levels. Through a re-analysis of ENCODE data, we show that replicability of transcript abundances will provide misleading estimates of the replicability of conditional variation in transcript abundances (i.e., most expression experiments). Heuristics which implicitly address this problem have emerged in quality control measures to obtain 'good' differential expression results. However, these methods involve strict filters such as discarding low expressing genes or using technical replicates to remove discordant transcripts, and are costly or simply ad hoc. As an alternative, we model gene-level replicability of differential activity using co-expressing genes. We find that sets of housekeeping interactions provide a sensitive means of estimating the replicability of expression changes, where the co-expressing pair can be regarded as pseudo-replicates of one another. We model the effects of noise that perturbs a gene's expression within its usual distribution of values and show that perturbing expression by only 5% within that range is readily detectable (AUROC~0.73). We have made our method available as a set of easily implemented R scripts. PMID:27082953

  3. AuPairWise: A Method to Estimate RNA-Seq Replicability through Co-expression

    PubMed Central

    Ballouz, Sara; Gillis, Jesse

    2016-01-01

    In addition to detecting novel transcripts and higher dynamic range, a principal claim for RNA-sequencing has been greater replicability, typically measured in sample-sample correlations of gene expression levels. Through a re-analysis of ENCODE data, we show that replicability of transcript abundances will provide misleading estimates of the replicability of conditional variation in transcript abundances (i.e., most expression experiments). Heuristics which implicitly address this problem have emerged in quality control measures to obtain ‘good’ differential expression results. However, these methods involve strict filters such as discarding low expressing genes or using technical replicates to remove discordant transcripts, and are costly or simply ad hoc. As an alternative, we model gene-level replicability of differential activity using co-expressing genes. We find that sets of housekeeping interactions provide a sensitive means of estimating the replicability of expression changes, where the co-expressing pair can be regarded as pseudo-replicates of one another. We model the effects of noise that perturbs a gene’s expression within its usual distribution of values and show that perturbing expression by only 5% within that range is readily detectable (AUROC~0.73). We have made our method available as a set of easily implemented R scripts. PMID:27082953

  4. IRcall and IRclassifier: two methods for flexible detection of intron retention events from RNA-Seq data

    PubMed Central

    2015-01-01

    Background The emergence of next-generation RNA sequencing (RNA-Seq) provides tremendous opportunities for researchers to analyze alternative splicing on a genome-wide scale. However, accurate detection of intron retention (IR) events from RNA-Seq data has remained an unresolved challenge in next-generation sequencing (NGS) studies. Results We propose two new methods: IRcall and IRclassifier to detect IR events from RNA-Seq data. Our methods combine together gene expression information, read coverage within an intron, and read counts (within introns, within flanking exons, supporting splice junctions, and overlapping with 5' splice site/ 3' splice site), employing ranking strategy and classifiers to detect IR events. We applied our approaches to one published RNA-Seq data on contrasting skip mutant and wild-type in Arabidopsis thaliana. Compared with three state-of-the-art methods, IRcall and IRclassifier could effectively filter out false positives, and predict more accurate IR events. Availability The data and codes of IRcall and IRclassifier are available at http://mlg.hit.edu.cn/ybai/IR/IRcallAndIRclass.html PMID:25707295

  5. aPPRove: An HMM-Based Method for Accurate Prediction of RNA-Pentatricopeptide Repeat Protein Binding Events.

    PubMed

    Harrison, Thomas; Ruiz, Jaime; Sloan, Daniel B; Ben-Hur, Asa; Boucher, Christina

    2016-01-01

    Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The [Formula: see text] class contains tandem [Formula: see text]-type motif sequences, and the [Formula: see text] class contains alternating [Formula: see text], [Formula: see text] and [Formula: see text] type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a [Formula: see text]-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the [Formula: see text] class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for [Formula: see text]-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~approve. PMID:27560805

  6. aPPRove: An HMM-Based Method for Accurate Prediction of RNA-Pentatricopeptide Repeat Protein Binding Events

    PubMed Central

    Harrison, Thomas; Ruiz, Jaime; Sloan, Daniel B.; Ben-Hur, Asa; Boucher, Christina

    2016-01-01

    Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The P class contains tandem P-type motif sequences, and the PLS class contains alternating P, L and S type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a PLS-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the PLS class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for PLS-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~approve. PMID:27560805

  7. A method for the large scale isolation of high transformation efficiency fungal genomic DNA.

    PubMed

    Zhang, D; Yang, Y; Castlebury, L A; Cerniglia, C E

    1996-12-01

    A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported. This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature. The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA. Sau3AI partially digested DNA showed high transformation efficiency (> 10(6)/100 ng DNA) when ligated to ZAP-express lambda vector. PMID:8961565

  8. Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus

    DOEpatents

    Lasure, Linda L [Fall City, WA; Dai, Ziyu [Richland, WA

    2008-10-21

    The invention includes isolated polynucleotide molecules that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention includes a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. The polynucleotide sequence is expressed to promote a first morphology. The first morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a second morphology.

  9. Comparative Evaluation of Commercially Available Manual and Automated Nucleic Acid Extraction Methods for Rotavirus RNA Detection in Stool

    PubMed Central

    Esona, Mathew D.; McDonald, Sharla; Kamili, Shifaq; Kerin, Tara; Gautam, Rashi; Bowen, Michael D.

    2015-01-01

    Rotaviruses are a major cause of viral gastroenteritis in children. For accurate and sensitive detection of rotavirus RNA from stool samples by reverse transcription-polymerase chain reaction (RT-PCR), the extraction process must be robust. However, some extraction methods may not remove the strong RT-PCR inhibitors known to be present in stool samples. The objective of this study was to evaluate and compare the performance of six extraction methods used commonly for extraction of rotavirus RNA from stool, which have never been formally evaluated: the MagNA Pure Compact, KingFisher Flex and NucliSENS® easyMAG® instruments, the NucliSENS® miniMAG® semi-automated system, and two manual purification kits, the QIAamp Viral RNA kit and a modified RNaid® kit. Using each method, total nucleic acid or RNA was extracted from eight rotavirus-positive stool samples with enzyme immunoassay optical density (EIA OD) values ranging from 0.176 to 3.098. Extracts prepared using the MagNA Pure Compact instrument yielded the most consistent results by qRT-PCR and conventional RT-PCR. When extracts prepared from a dilution series were extracted by the 6 methods and tested, rotavirus RNA was detected in all samples by qRT-PCR but by conventional RT-PCR testing, only the MagNA Pure Compact and KingFisher Flex extracts were positive in all cases. RT-PCR inhibitors were detected in extracts produced with the QIAamp Viral RNA Mini kit. The findings of this study should prove useful for selection of extraction methods to be incorporated into future rotavirus detection and genotyping protocols. PMID:24036075

  10. Isolation of human dermis derived mesenchymal stem cells using explants culture method: expansion and phenotypical characterization.

    PubMed

    Park, Jeong-Ran; Kim, Eunjeong; Yang, Jungwon; Lee, Hanbyeol; Hong, Seok-Ho; Woo, Heung-Myong; Park, Sung-Min; Na, Sunghun; Yang, Se-Ran

    2015-06-01

    Recent studies have reported that stem cells can be isolated from a wide range of tissues including bone marrow, fatty tissue, adipose tissue and placenta. Moreover, several studies also suggest that skin dermis could serve as a source of stem cells, but are of unclear phenotype. Therefore, we isolated and investigated to determine the potential of stem cell within human skin dermis. We isolated cells from human dermis, termed here as human dermis-derived mesenchymal stem cells (hDMSCs) which is able to be isolated by using explants culture method. Our method has an advantage over the enzymatic method as it is easier, less expensive and less cell damage. hDMSCs were maintained in basal culture media and proliferation potential was measured. hDMSCs were highly proliferative and successfully expanded with no additional growth factor. In addition, hDMSCs revealed normal karyotype and expressed high levels of CD90, CD73 and CD105 while did not express the surface markers for CD34, CD45 and HLA-DR. Also, we confirmed that hDMSCs possess the capacity to differentiate into multiple lineage including adipocyte, osteocyte, chondrocyte and precursor of hepatocyte lineage. Considering these results, we suggest that hDMSCs might be a valuable source of stem cells and could potentially be a useful source of clinical application. PMID:25163610

  11. A Standardized Method of Isolating Adipose-Derived Stem Cells for Clinical Applications.

    PubMed

    Raposio, Edoardo; Caruana, Giorgia; Petrella, Maira; Bonomini, Sabrina; Grieco, Michele P

    2016-01-01

    White adipose tissue is the most abundant and accessible source of stem cells in the adult human body. In this paper, we present a standardised and safe method of isolating and maximizing the number of adipose-derived stem cells (ASCs) from conventional liposuction for clinical applications, which was carried out through both mechanical (centrifuge) and enzymatic (collagenase) means. Isolated cells were characterized through flow cytometry assay. Gathered data showed a greater amount (9.06 × 10(5) ASCs from 100 mL of adipose tissue) of isolated ASCs compared to previous protocol, also with high (99%) cell vitality; the procedure we presented is easy and fast (80 minutes), allowing collecting a significative number of mesenchymal stem cells, which can be used for clinical purposes, such as wound healing. PMID:26418805

  12. Rapid Method for Isolation of Desiccation-Tolerant Strains and Xeroprotectants▿

    PubMed Central

    Narváez-Reinaldo, J. J.; Barba, I.; González-López, J.; Tunnacliffe, A.; Manzanera, M.

    2010-01-01

    A novel biotechnological process has been developed for the isolation of desiccation-tolerant microorganisms and their xeroprotectants, i.e., compatible solutes involved in long-term stability of biomolecules in the dry state. Following exposure of soil samples to chloroform, we isolated a collection of desiccation-tolerant microorganisms. This collection was screened for the production of xeroprotectants by a variation of the bacterial milking (osmotic downshock) procedure and by a novel air-drying/rehydration (“dry milking”) incubation method. The resultant solutes were shown to protect both proteins and living cells against desiccation damage, thereby validating them as xeroprotectants. Nuclear magnetic resonance (NMR) analytical studies were performed to identify the xeroprotectants; synthetic mixtures of these compounds were shown to perform similarly to natural isolates in drying experiments with proteins and cells. This new approach has biotechnological and environmental implications for the identification of new xeroprotectants of commercial and therapeutic value. PMID:20562279

  13. A novel isolation method for hen egg yolk antibody, "IgY".

    PubMed

    Hatta, H; Kim, M; Yamamoto, T

    1990-10-01

    A method for isolation of egg yolk immunoglobulin, IgG, a livetin protein, was investigated. Several natural gums (carrageenan and xanthan gum) were found to be effective for removal of yolk lipoprotein as a precipitate. The effect was pronounced with lambda-carrageenan and the lipid content in the supernatant after removal of the resulting precipitate was less than 0.4% of that of egg yolk. IgY remained in this supernatant, with a yield of 86%, and was isolated by chromatography on a column of DEAE-Sephacel followed by salting-out with sodium sulfate. IgY thus isolated was almost pure (98%) and the yield was 70 to 100 mg per egg. PMID:1368596

  14. Extending lock-in methods: term isolation detection of nonlinear signals.

    PubMed

    Jarvis, Thomas W

    2016-08-01

    We show that components of a nonlinear signal can be measured using phase-sensitive detection at unconventional demodulation frequencies, allowing us to isolate individual terms from the signal. To demonstrate this technique, autocorrelation measurements of an ultrafast pulsed laser were performed using two-photon absorption. In this example, the isolation of individual autocorrelation terms may provide internal consistency checks to improve the precision and accuracy of pulse characterization. More generally, this scheme can be extended to a range of nonlinear measurements. As a demonstration, we analyze a three-photon autocorrelation model, showing that many nonlinear signals can be studied with this method. We anticipate that term isolation detection will find application in a broad range of experiments, such as multidimensional Fourier transform spectroscopy or coherent anti-Stokes Raman spectroscopy. PMID:27505362

  15. A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

    PubMed Central

    Neuman, Joshua C.; Truchan, Nathan A.; Joseph, Jamie W.; Kimple, Michelle E.

    2014-01-01

    Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression. PMID:24998772

  16. A method for mouse pancreatic islet isolation and intracellular cAMP determination.

    PubMed

    Neuman, Joshua C; Truchan, Nathan A; Joseph, Jamie W; Kimple, Michelle E

    2014-01-01

    Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3(H)]-thymidine incorporation, protein abundance, and mRNA expression. PMID:24998772

  17. Inner structural vibration isolation method for a single control moment gyroscope

    NASA Astrophysics Data System (ADS)

    Zhang, Jingrui; Guo, Zixi; Zhang, Yao; Tang, Liang; Guan, Xin

    2016-01-01

    Assembling and manufacturing errors of control moment gyros (CMG) often generate high frequency vibrations which are detrimental to spacecrafts with high precision pointing requirement. In this paper, some design methods of vibration isolation between CMG and spacecraft is dealt with. As a first step, the dynamic model of the CMG with and without supporting isolation structures is studied and analyzed. Subsequently, the frequency domain analysis of CMG with isolation system is performed and the effectiveness of the designed system is ascertained. Based on the above studies, an adaptive design suitable with appropriate design parameters are carried out. A numerical analysis is also performed to understand the effectiveness of the system and the comparison made. The simulation results clearly indicate that when the ideal isolation structure was implemented in the spacecraft, the vibrations generated by the rotor were found to be greatly reduced, while the capacity of the output torque was not lost, which means that the isolation system will not affect the performance of attitude control.

  18. Isolation of Bartonella (Rochalimaea) henselae: effects of methods of blood collection and handling.

    PubMed Central

    Brenner, S A; Rooney, J A; Manzewitsch, P; Regnery, R L

    1997-01-01

    Bartonella (Rochalimaea) henselae causes cat-scratch disease, bacillary angiomatosis, peliosis hepatis, and fever in humans. B. henselae can be difficult to culture axenically, and as many as 5 weeks may be required before colonies are visible. We compared how different methods of blood collection and handling affect isolation of this pathogen. Blood specimens from B. henselae-infected cats were collected in both EDTA and Isolator blood-lysis tubes and were subsequently plated onto rabbit blood-brain heart infusion agar by using three different schedules: plating immediately, plating after 24 h at 25 degrees C, and plating after 26 days at -65 degrees C. Colonies were counted 14 and 35 days after plating. Blood collected in tubes containing EDTA, frozen at -65 degrees C, and then plated on blood agar yielded a median of 60,000 CFU/ml, compared with 25,333 CFU/ml after collection in the Isolator tubes (P < 0.01). Frozen blood yielded the largest number of B. henselae colonies for any of the schedules tested. These results support previous observations that the Isolator system is more sensitive than tubes containing EDTA for isolation of B. henselae and suggest that, for cat blood, collection in tubes containing EDTA and subsequent freezing may further improve the sensitivity of detection of B. henselae. PMID:9041385

  19. Application of retrograde dissection method for isolation of bone marrow cells from rat femurs and tibiae.

    PubMed

    Li, C M; Fu, B M; Zhang, L C; Tang, B; Zhu, L; Zhao, Y; Zhang, J

    2016-01-01

    Currently, there is no practical and efficient method for the isolation of bone marrow cells (BMCs) from rat femurs and tibiae. Here, we attempted to develop a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae. Rat femurs and tibiae were dissected from the ankle to the hip joint; subsequently, a three-step "locate-slide-twist" procedure was performed using scissors and forceps to remove the femurs and tibiae completely, from the surrounding musculature. The bones were flushed with phosphate-buffered saline to harvest BMCs. The femurs and tibiae were dissected in 1.8 ± 0.6 min, and the BMC suspension preparation time was 13.1 ± 2.3 min. The bone marrow cavities did not incur any fractures or injuries during the isolation. Culture of harvested BMCs for 72 h led to a significant increase in cell number from 4.4 ± 0.3 x 106 to 6.9 ± 0.7 x 10(6) (P < 0.01) with no significant decrease in viability (98.1 ± 0.6% vs 96.2 ± 1.1%; P > 0.05). Microscopic examination of the isolated BMCs after the 72-h incubation period revealed the no-microbial or muscle cell contamination. Furthermore, flow cytometry revealed that cultured BMCs (72-h culture) grew well. Here, we have reported a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae by using retrograde dissection. This method can be used to harvest a large number of viable BMCs without the risk of contamination from muscle and connective tissues. PMID:27323101

  20. Molecular characterization of Staphylococcus aureus isolates from southwest of Iran using spa and SCCmec typing methods.

    PubMed

    Darban-Sarokhalil, Davood; Khoramrooz, Seyed Sajjad; Marashifard, Masoud; Malek Hosseini, Seyed Ali Asghar; Parhizgari, Najmeh; Yazdanpanah, Mahboobeh; Gharibpour, Farzaneh; Mirzaii, Mehdi; Sharifi, Bahman; Haeili, Mehri

    2016-09-01

    Staphylococcus aureus remains a major cause of nosocomial infection worldwide. Characterization of S. aureus isolates circulating in the southwest of Iran will contribute to understand and control the spread of the strains in this area. spa and SCCmec typing methods were used for genotyping of 125 S. aureus isolates obtained from two teaching hospitals in Ahvaz. Drug susceptibility testing was performed by using disk diffusion method. Frequency of the methicillin resistant S. aureus (MRSA) isolates was 39% (n = 34) and 27% (n = 10) in Emam Khomeini and Golestan hospi