【摘要】 目的筛选合适的β-肌动蛋白(β-actin)引物以排除RNA样本中残存的DNA对人类基因表达分析的干扰。方法利用EMBL、PSEUDOGENE等生物信息数据库,寻找β-actin的假基因。同时自行设计和从国内外文献中筛选了众多β-actin引物。选用其中具有代表性的5对跨过内含子的引物,分别将基因组DNA、未经逆转录的RNA、cDNA作为模板,对这5对引物进行聚合酶链反应(PCR)扩增。结果生物信息学分析发现β-actin有37个假基因并且发现绝大多数的研究者所使用的引物都存在假基因干扰问题。当分别用DNA和cDNA作为模板时,5对引物中的1对跨过内含子的引物能扩增出大小不同的条带。结论使用该对引物能有效发现和排除RNA样本中所残存的DNA的干扰,令人类基因表达分析得出更准确的结果。更多还原

【Abstract】 Objective Suitable β-actin primes are screened and selected to avoid the interference of β-actin processed pseudogenes in the human gene expression analysis.Methods β-actin pseudogenes were found out with bioinformatics databases such as EMBL, PSEUDOGENE. Many primers were designed or selected referring to different literatures. Five pairs of β-actin primers were tested by comparing with the PCR results using DNA, nonreversed transcribed RNA, cDNA as templates. Results β-actin gene had 37 pseudogenes and many primers used by most researchers had the problem of being interfered by psedudogenes. One pair of primers could amplify different sizes of products respectively when using DNA and cDNA as templates. Conclusion It is concluded that, with this pair of primers, the interference of contaminating genomic DNA can be easily found out and aboided, thus a much accurate result of human gene expression analysis can be obtained.更多还原