用于小分子筛选测定的高通量逆转录聚合酶链反应
High-Throughput RT-PCR for small-molecule screening assays.
作者信息
Bittker Joshua A
机构信息
Chemical Biology Platform, Broad Institute of MIT and Harvard, Cambridge, MA 617-714-7373.
出版信息
Curr Protoc Chem Biol. 2012 Mar 1;4(1):49-63. doi: 10.1002/9780470559277.ch110204.
Abstract
Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.
摘要
长期以来，使用实时逆转录聚合酶链反应（RT-PCR）对mRNA表达水平进行定量测量一直用于分析感兴趣的组织或细胞系中的表达差异。该方法在测量因小分子或siRNA等干扰物导致的基因表达变化方面使用频率略低。新型液体处理和实时PCR分析仪器的出现，以及RT-PCR全程试剂盒的商业供应，使得该方法能够用于高通量小分子筛选，其规模可与传统高通量筛选（HTS）分析相媲美。本方案重点关注将定量RT-PCR用作主要小分子筛选分析所需的特殊注意事项，包括mRNA分离和分析的可用不同方法。
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