摘要
目的:研究肝素在逆转录过程中的作用。方法:提取人肺胚胎二倍体细胞和肝素抗凝血中总RNA,经1%琼脂糖凝胶电泳鉴定RNA的质量好后,逆转录合成相应的cDNA,分别进行看家基因β-actin的聚合酶链反应(PCR),其产物用1%琼脂糖凝胶电泳检测;用基因芯片技术检测cDNA。将肝素抗凝血中提取的总RNA分为等量的两部分,分别用肝素酶或氯化锂处理RNA进行逆转录为cDNA和RNA合成cDNA后再用肝素酶或氯化锂处理,分别进行看家基因β-actin的聚合酶链反应(PCR),其产物用1%琼脂糖凝胶电泳检测;并用基因芯片技术检测氯化锂处理RNA后cDNA。结果:二倍体细胞来源的RNA为模板,能扩增出β-actin产物,并且在基因芯片上有杂交点。肝素抗凝血来源的RNA,在肝素酶或氯化锂作用后行逆转录者,能扩增到β-actin的目的片段,且基因芯片上有杂交点;而在未处理者不能扩增到目的片段,且基因芯片上无杂交点。结论:肝素是一种逆转录的抑制剂,对RNA逆转录cDNA过程有较强的抑制作用。
Objective：To investigate heparin＇s function in the course of reverse-transcription.Methods：Total RNAs were isolated from human embryonic lung diploid cells and heparinized clinical specimens bloods by Trizol method,which the quality of RNAs were confirmed by 1% agarose gel electrophoresis,and reverse-transcripted their corresponding cDNAs.Then,keeping-geneβ-actin products,with polymerase chain reaction（PCR）,which were detected by 1% agarose gel electrophoresis;the existence of their corresponding cDNAs was detected with gene chip technology.In addition,partly total RNAs isolated from heparinized clinical specimens bloods were divided into two parts,one of which were pretreated with heprinase or lithium chloride,another of which were treated after their cDNA with heprinase or lithium chloride.Their cDNA and corresponding keeping-geneβ-actin products were detected with gene chip technology or PCR after two treations,.Results：RNAs isolated from human embryonic lung diploid cells could be amplified toβ-actin productions and their cDNAs could be intercrossed on microarray.RNAs isolated from heparinized clinical specimens,which were pretreated with heprinase or lithium chloride,could be detected like RNAs isolated from cells,but RNAs which were not pretreated with heprinase or lithium chloride could not.Conclusion：Heparin is an inhibitor of reverse transcription,and has strong inhibition during reverse transcription from RNA to cDNA.
出处
《现代生物医学进展》
CAS
2008年第8期1508-1510,共3页
Progress in Modern Biomedicine
基金
昆明市科技计划项目(04S06012-01)
