RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

An RT-PCR Method of Excluding the Interference of Pseudogene HUTP14C on the Detection of HUTP14A mRNA

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT-PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3'-UTR的siRNA沉默HUTP14C mRNA后,再用RT-PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT-PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3'-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3'-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。

Human U three protein 14C(HUTP14C)is a pseudogene of human U three protein 14A(HUTP14A).The sequences of their transcripts are 95%conserved.Existence of HUTP14C may cause experimental error when the conventional RT-qPCR is used to evaluate the abundance of HUTP14A mRNA.The aim of this study is to establish an accurate RT-PCR procedure for the evaluation of HUTP14A mRNA.We have designed primers that can specifically amplify HUTP14A or HUTP14C fragments from human genomic DNA or cDNA,respectively.An RT-PCR procedure was generated to exclude the interference of the pseudogene HUTP14C on the detection of HUTP14A mRNA.The mRNA of HUTP14A and HUTP14C was detected in 18 pairs of hepatocellular cancer tissues.When detecting the mRNA levels of functional genes that possess pseudogenes,PCR should be directly performed using the total RNA extracted from cells or tissues after DNaseⅠtreatment to exclude the possibility of DNA contamination.To exclude the influence of pseudogene mRNA on the evaluation of the mRNA of functional gene,specific siRNAs targeting the 3'-UTR region of the pseudogene can be used to silence the mRNA of the pseudogene before RT-PCR is performed.When detecting the mRNA levels of functional genes in pathological tissues,the specific primers of functional genes can be designed according to the sequence differences between the pseudogenes and their functional genes.The primers for pseudogenes can be designed according to the longer 3'-UTR region of pseudogenes.Thereby,the contaminations of pseudogenes can be excluded when detecting transcription levels of the functional genes by RT-qPCR.