Loudig Olivier, Milova Ekaterina, Brandwein-Gensler Margaret, Massimi Aldo, Belbin Thomas J, Childs Geoffrey, Singer Robert H, Rohan Thomas, Prystowsky Michael B
Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, USA.
Nucleic Acids Res. 2007;35(15):e94. doi: 10.1093/nar/gkm510. Epub 2007 Jul 17.
Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35-41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods.
对来自已完成的临床试验和常规临床护理的福尔马林固定石蜡包埋(FFPE)样本进行基因表达谱分析,有可能产生将基因与临床参数相关联并建立联系的有价值信息。为了从高度片段化的FFPE-RNA制备高质量的cDNA(此前无法进行高通量分析),我们设计了一种基于在T7体外转录(IVT)扩增之前恢复不完整cDNA序列的核酸修复的新策略。我们将此策略描述为互补模板逆转录(CT-RT),因为从FFPE-RNA获得的短单链T7-寡聚-dT24-VN-DNA序列用作正义RNA文库中包含的互补RNA模板逆转录的引物。我们通过确定匹配的10岁冷冻和FFPE乳腺癌样本的表达谱之间的相关性来验证我们的检测方法。我们表明,通过CT-RT恢复的cDNA转录本的T7 IVT扩增是一个特定且可靠的过程,能够恢复通过直接对FFPE-RNA进行T7 IVT扩增无法检测到的转录特征。此外,与匹配的冷冻组织相比,CT-RT从存档的乳腺和宫颈样本中恢复了35-41%的转录本;并且这些谱包括组织特异性转录本。我们的结果表明,CT-RT允许对以前方法无法分析的严重降解的RNA进行微阵列分析。