Detection of Antisense RNA Transcripts by Strand-Specific RT-PCR
Detection of Antisense RNA Transcripts by Strand-Specific RT-PCR
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DOI：
10.1007/978-1-60761-629-0_9
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发表时间：
2010-01-01
期刊：
影响因子：
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通讯作者：
Saville, Barry J.
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文献类型：
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作者：
Ho, Eric C. H.;Donaldson, Michael E.;Saville, Barry J.
全面的 gnome 注释需要广泛的 cDNA 分析。该分析鉴定了许多不同真核生物中的天然反义转录本 (NAT)，它们与 microRNA、siRNA 和 piRNA 不同。这种广泛的保护支持 NAT 在调节细胞过程中发挥重要作用的可能性。研究它们的作用需要确认表达序列标签 (EST) 数据并检测不同细胞背景中的反义转录本。本章介绍了使用逆转录聚合酶链式反应 (RT-PCR) 方法检测反义转录本。该方案旨在通过利用反义定向引物和寡聚 dT 来引发第一链合成，从而减少反义转录物筛选过程中第一链合成反应的数量。在第一链合成中使用有义和反义定向引物进一步证实了这些结果。结果表明，筛选的优化需要适当的控制，以确认 gDNA 污染的去除并排除自引发作为第一链产物的来源。
Comprehensive gnome annotation requires extensive cDNA analysis. This analysis has identified natural antisense transcripts (NATs), which are distinct from the microRNAs, siRNAs, and piRNAs, in a number of diverse eukaryotes. This wide conservation supports the possibility of an important role for NATs in regulating cellular processes. Investigating their roles requires the confirmation of expressed sequence tag (EST) data and the detection of antisense transcripts in distinct cellular backgrounds. This chapter describes the use of a reverse transcription polymerase chain reaction (RT-PCR) method for the detection of antisense transcripts. The protocol was designed to reduce the number of first strand synthesis reactions during screening for antisense transcripts through the utilization of antisense directed primers and oligo dT to prime first strand synthesis. These results are further confirmed using sense and antisense directed primers in first strand synthesis. Results indicate that optimization of the screens requires proper controls to confirm removal of gDNA contamination and to rule out self-priming as a source of first strand products.