Yang Wen, Maqsodi Botoul, Ma Yunqing, Bui Son, Crawford Kimberly L, McMaster Gary K, Witney Frank, Luo Yuling
Genospectra, Inc., Fremont, CA 94555, USA.
Biotechniques. 2006 Apr;40(4):481-6. doi: 10.2144/000112133.
Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.
福尔马林固定、石蜡包埋（FFPE）组织是用于基因表达谱分析的重要存档材料来源。我们在此报告一种改良的分支DNA分析方法的开发，该方法可直接定量FFPE组织切片匀浆中的信使RNA（mRNA）转录本，无需进行RNA分离和逆转录成cDNA。福尔马林固定对分支DNA分析基本没有影响，RNA降解只会使信号略微降低2至3倍。在相同条件下，福尔马林固定和RNA降解会大大降低实时逆转录PCR（RT-PCR）效率，分别使信号降低多达15倍和1400倍。尽管这两种技术都可以从FFPE人肺肿瘤标本中生成具有生物学意义的表达谱，但在测试条件下，分支DNA分析比实时RT-PCR更敏感。因此，我们的结果表明，分支DNA分析是存档组织中基因表达回顾性分析的理想工具。
