摘要
该文建立了高效肝组织及细胞总RNA抽提、反转录以进行基因克隆和实时定量PCR(q-RT-PCR)的方法。比较了2种反转录酶(M-MLV和SuperScriptII)、2种cDNA合成引物(OligodT和random 6 primer)对总RNA反转录效率的影响,与4种DNA聚合酶(Taq聚合酶、Pfu聚合酶、LAtaq聚合酶、Prime Star聚合酶)进行长片段基因克隆的能力及效率;同时,该研究比较了不同质量总RNA对有效进行q-RT-PCR与长片段分子克隆的影响。新建立的RNA提取方法使得RNA完整性和均一性提高。RNA的完整性及均一性对长片段cDNA的克隆至关重要。部分降解的组织RNA及细胞RNA仅适合于q-RT-PCR检测mRNA的表达水平,而不适合于cDNA的克隆。
In this report, we have developed efficient methods for total RNA extraction from hepatocarcinoma tissues and cell lines, reverse transcription, cDNA cloning and quantitive real-time PCR （q-RT-PCR）. We compared the effect of two reverse transcriptase （M-MLV, SuperScriptll） on the efficiency of total RNA reverse transcription and four DNA polymerase （Taq polymerase, Pfu polymerase, LA taq polymerase, Prime Star polymerase） in cloning large fragment of cDNA. The impact of different RNA integrity on q-RT-PCR and large fragment of cDNA cloning had also been investigated in this study. The newly developed RNA extraction method greatly improved the integrity of total RNA. We found that the integrity of total RNA is vital to large fragment cDNA cloning. RNA degraded at some extent is only suitable for q-RT-PCR analysis but not for cDNA cloning.
基金
国家自然科学基金(30800407)
关键词
肝癌组织及细胞系
总RNA抽提
反转录
Q-RT-PCR
长片段基因克隆
Hepatocarcinoma tissues and cell lines
Total RNA extraction
Reverse transcription
q-RT-PCR
Cloning of large fragment cDNA
