Li J J, Domann F, Oberley L W
Radiation Research Laboratory, University of Iowa, Iowa City 52242, USA.
Biochem Biophys Res Commun. 1995 Nov 13;216(2):610-8. doi: 10.1006/bbrc.1995.2666.
We report here a convenient RT-PCR method to distinguish plasmid human MnSOD cDNA transcripts from the endogenous MnSOD gene products without engineering the cDNA insert. When a specific antisense primer for the carrier vector sequence was paired with a sense primer for the human MnSOD cDNA in RT-PCR analysis, a unique amplicon with the expected size was generated in MnSOD cDNA transfected cells but not in the wild type or vector control cells. The same primers were also used in genomic DNA-PCR to demonstrate genomic incorporation of cDNA in stably transfected cells. This method is convenient and specific in determining exogenous cDNA incorporation and expression in transfectants especially when transcripts of cDNA are difficult to separate from the endogenous mRNA by other methods.
我们在此报告一种简便的逆转录聚合酶链反应（RT-PCR）方法，无需对cDNA插入片段进行改造，就能区分质粒人锰超氧化物歧化酶（MnSOD）cDNA转录本与内源性MnSOD基因产物。在RT-PCR分析中，当用于载体序列的特异性反义引物与用于人MnSOD cDNA的正义引物配对时，在转染了MnSOD cDNA的细胞中会产生具有预期大小的独特扩增子，而在野生型或载体对照细胞中则不会产生。相同的引物也用于基因组DNA-PCR，以证明cDNA在稳定转染细胞中的基因组整合。该方法在确定转染子中外源cDNA的整合和表达方面既方便又特异，尤其是当cDNA转录本难以通过其他方法与内源性mRNA分离时。
