Smith R D, Ogden C W, Penny M A
Imperial College of Science, Technology and Medicine, London, UK.
Biotechniques. 2001 Oct;31(4):776-8, 780, 782. doi: 10.2144/01314st03.
Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.
RNA样本中的基因组DNA污染对RT-PCR有重要影响，特别是如果存在与所研究基因相关的假基因时，因为来自假基因的扩增和逆转录的cDNA很难区分。去除DNA污染的方法无法保证在不损失RNA数量或质量的情况下样本中绝对不存在DNA，而这对于少量RNA或低表达水平转录本的研究可能至关重要。在此，我们描述了一种RT-PCR通用技术，该技术将一个不存在于基因组DNA中的序列应用于逆转录cDNA的5'末端，并利用此序列使反向PCR引物退火，以排除未修饰RNA样本中的基因组DNA扩增。
