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Hengphon Too - One of the best experts on this subject based on the ideXlab platform.
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direct quantification of mrna and mirna from Cell Lysates using reverse transcription real time pcr a multidimensional analysis of the performance of reagents and workflows
PLOS ONE, 2013Co-Authors: Hengphon TooAbstract:Substantial efforts have been devoted to in vitro testing of candidate chemotherapeutics by profiling transcriptional changes across the collection of NCI-60 Cell-lines. A work-flow with reagents that enable the direct quantification of RNA of different molecular sizes simultaneously in the same sample without laborious total RNA isolation will invariably increase the throughput and accuracy of the study. MicroRNAs (miRNAs) are known to regulate most Cellular functions, acting post-transcriptionally by repressing numerous eukaryotic mRNAs. Recent findings on the remarkable stability of miRNA prompted us to investigate the feasibility of quantifying the expression levels of both mRNA and miRNA directly from Cell Lysates (Cell-to-Ct). Multidimensional analyses of the expressions of mRNA and miRNA across seven NCI-60 Cell lines and multiple reagents were conducted to assess the performances of these reagents and workflows for Cell-to-Ct measurements using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Quantification of RNA species using Lysates prepared from an in-house and one of the commercial reagents demonstrated comparable performance to those prepared by the more laborious and conventional method of using guanidinium-phenol-chloroform. Additionally, miRNA was found to be highly stable in the Cell Lysates when incubated at room temperature for prolonged period of time and subjected to multiple freeze-thaw cycles. In summary, this study demonstrated significant differences in pre-analytical performance of a variety of commercially available reagents and described a cost-effective reagent useful for rapid, scalable, and high-throughput workflow for the detection of mRNA and miRNA from the same biological sample.
Haruhiko Murata - One of the best experts on this subject based on the ideXlab platform.
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Endogenous RNase inhibitor contributes to stability of RNA in crude Cell Lysates: Applicability to RT-qPCR.
Analytical biochemistry, 2016Co-Authors: Xiao Wang, Belete Teferedegne, Kenneth Shatzkes, Haruhiko MurataAbstract:Abstract Crude Cell Lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial Cell-lysis reagents for the preparation of RT-qPCR-ready Cell Lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in Cell Lysates either freshly prepared or previously stored frozen at −80 °C. In the present study, we have demonstrated that the stability of RNA observed in our Cell Lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating Lysates prepared from Cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating Cell Lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.
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A simple, inexpensive method for preparing Cell Lysates suitable for downstream reverse transcription quantitative PCR
Scientific reports, 2014Co-Authors: Kenneth Shatzkes, Belete Teferedegne, Haruhiko MurataAbstract:Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude Cell Lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready Cell Lysates from MDCK Cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37°C incubation for 1–2 hours, in Cell-lysate samples either freshly prepared or previously stored frozen at −80°C.
Kenneth Shatzkes - One of the best experts on this subject based on the ideXlab platform.
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Endogenous RNase inhibitor contributes to stability of RNA in crude Cell Lysates: Applicability to RT-qPCR.
Analytical biochemistry, 2016Co-Authors: Xiao Wang, Belete Teferedegne, Kenneth Shatzkes, Haruhiko MurataAbstract:Abstract Crude Cell Lysates are increasingly used as input for direct analysis by reverse transcription quantitative PCR (RT-qPCR), particularly for high-throughput applications. We previously demonstrated that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercial Cell-lysis reagents for the preparation of RT-qPCR-ready Cell Lysates; addition of an exogenous RNase inhibitor (RI) to the lysis buffer was found to be unnecessary to maintain RNA stability in Cell Lysates either freshly prepared or previously stored frozen at −80 °C. In the present study, we have demonstrated that the stability of RNA observed in our Cell Lysates is due to the presence of the endogenous RI. Furthermore, we have established the generalizability and applicability of this phenomenon by evaluating Lysates prepared from Cell lines commonly used in virology (A549, HeLa, MDCK, and Vero). Awareness of the mechanism underlying RNA stability may engender greater confidence in generating Cell Lysates for RT-qPCR without relying on addition of exogenous RI (a substantial cost-saving benefit) and encourage appropriate practices for handling and storage of samples.
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A simple, inexpensive method for preparing Cell Lysates suitable for downstream reverse transcription quantitative PCR
Scientific reports, 2014Co-Authors: Kenneth Shatzkes, Belete Teferedegne, Haruhiko MurataAbstract:Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude Cell Lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready Cell Lysates from MDCK Cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37°C incubation for 1–2 hours, in Cell-lysate samples either freshly prepared or previously stored frozen at −80°C.
Risto Renkonen - One of the best experts on this subject based on the ideXlab platform.
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analysis of nucleotide sugars from Cell Lysates by ion pair solid phase extraction and reversed phase high performance liquid chromatography
Glycoconjugate Journal, 2001Co-Authors: Jarkko Rabina, Minna Maki, E Savilahti, Nina Jarvinen, Leena Penttila, Risto RenkonenAbstract:Analysis of nucleotide sugar metabolism is essential in studying glycosylation in Cells. Here we describe practical methods for both extraction of nucleotide sugars from Cell Lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial Cells are described here as specific applications.
Elwyn Y. Loh - One of the best experts on this subject based on the ideXlab platform.
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Competitor mRNA fragments for quantitation of cytokine specific transcripts in Cell Lysates
Molecular immunology, 1993Co-Authors: Danuta Kozbor, Elizabeth Hyjek, Richard Wladerkiewicz, Zhien Wang, Martin Wang, Elwyn Y. LohAbstract:Synthetic RNAs (sRNAs) specific for four human cytokines were constructed and used as an exogenous internal standard in a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The sequences of the sRNA and the target mRNA were identical except for a duplication or deletion of approximately 100 nucleotides. The size difference between these two templates permitted easy electrophoretic separation of their PCR products. The sRNA has polyadenylated sequences at the 3' end and can be added directly either to a Cell lysate before RNA purification or to a reverse transcription reaction. One pair of primers is used to amplify the internal standard and the target simultaneously, and the ratio of the two PCR products remains constant throughout the amplification. This technique can be applied to quantitate specific mRNA in as few as 10 Cells when the exogenous control is added directly to Cell Lysates. This method is sensitive, accurate and adaptable for quantitation of other transcripts.