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.
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.
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DOI：
10.1371/journal.pone.0072463
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Too HP
中科院分区：
文献类型：
--
作者：
Ho YK;Xu WT;Too HP
通过分析 NCI-60 细胞系集合中的转录变化，我们投入了大量精力来对候选化疗药物进行体外测试。使用试剂的工作流程能够同时直接定量同一样品中不同分子大小的 RNA，而无需费力地分离总 RNA，这必然会提高研究的通量和准确性。众所周知，MicroRNA (miRNA) 可以调节大多数细胞功能，通过抑制大量真核生物 mRNA 在转录后发挥作用。最近关于 miRNA 卓越稳定性的发现促使我们研究直接从细胞裂解物（细胞到 Ct）定量 mRNA 和 miRNA 表达水平的可行性。对 7 个 NCI-60 细胞系和多种试剂的 mRNA 和 miRNA 表达进行多维分析，以评估这些试剂的性能以及使用逆转录定量聚合酶链反应 (RT-qPCR) 进行细胞到 Ct 测量的工作流程。使用由内部试剂和一种商业试剂制备的裂解物对 RNA 种类进行定量，结果表明其性能与使用胍-苯酚-氯仿的更费力且更传统的方法制备的结果相当。此外，当在室温下长时间孵育并经历多次冻融循环时，miRNA 在细胞裂解物中高度稳定。总之，本研究证明了各种市售试剂的分析前性能存在显着差异，并描述了一种经济高效的试剂，可用于快速、可扩展和高通量的工作流程，用于检测同一生物样品中的 mRNA 和 miRNA。
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.
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影响因子：
5.7
作者：
Liu H;D'Andrade P;Fulmer-Smentek S;Lorenzi P;Kohn KW;Weinstein JN;Pommier Y;Reinhold WC
通讯作者：
Reinhold WC
影响因子：
5.7
作者：
Blower, Paul E.;Verducci, Joseph S.;Sadee, Wolfgang
通讯作者：
Sadee, Wolfgang
DOI：
10.1016/j.bbrc.2006.12.135
发表时间：
2007-02-23
影响因子：
3.1
作者：
Bravo, Valia;Rosero, Samuel;Pastori, Ricardo L.
通讯作者：
Pastori, Ricardo L.
影响因子：
2.6
作者：
Muyal, Jai Prakash;Muyal, Vandana;Fehrenbach, Heinz
通讯作者：
Fehrenbach, Heinz
影响因子：
1.2
作者：
Moltzahn, Felix;Hunkapiller, Nathan;Blelloch, Robert
通讯作者：
Blelloch, Robert