Volume 30 Issue 5
Sep.  2009
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YI Bin, CHANG Hong, CAO Yi. Improvement of Methods for Total RNA Extraction from Hepatocarcinoma Tissues and Cell Lines and Comparison of Reverse Transcription and cDNA Cloning Strategies. Zoological Research, 2009, 30(5): 520-526. doi: 10.3724/SP.J.1141.2009.05520
Citation: YI Bin, CHANG Hong, CAO Yi. Improvement of Methods for Total RNA Extraction from Hepatocarcinoma Tissues and Cell Lines and Comparison of Reverse Transcription and cDNA Cloning Strategies. Zoological Research, 2009, 30(5): 520-526. doi: 10.3724/SP.J.1141.2009.05520

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

doi: 10.3724/SP.J.1141.2009.05520
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  • Corresponding author: CAO Yi
  • Received Date: 2009-04-30
  • Rev Recd Date: 1900-01-01
  • Publish Date: 2009-10-22
  • 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.
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    沈阳化工大学材料科学与工程学院 沈阳 110142

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

doi: 10.3724/SP.J.1141.2009.05520
    Corresponding author: CAO Yi

Abstract: 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.

YI Bin, CHANG Hong, CAO Yi. Improvement of Methods for Total RNA Extraction from Hepatocarcinoma Tissues and Cell Lines and Comparison of Reverse Transcription and cDNA Cloning Strategies. Zoological Research, 2009, 30(5): 520-526. doi: 10.3724/SP.J.1141.2009.05520
Citation: YI Bin, CHANG Hong, CAO Yi. Improvement of Methods for Total RNA Extraction from Hepatocarcinoma Tissues and Cell Lines and Comparison of Reverse Transcription and cDNA Cloning Strategies. Zoological Research, 2009, 30(5): 520-526. doi: 10.3724/SP.J.1141.2009.05520

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