Improvement of Methods for Total RNA Extraction from Hepatocarcinoma Tissues and Cell Lines and Comparison of Reverse Transcription and cDNA Cloning Strategies

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, SuperScriptII) 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.

Keywords
Hepatocarcinoma tissues and cell lines; Total RNA extraction; Reverse transcription; q-RT-PCR; Cloning of large fragment cDNA