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Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis
Sean T Glenn1, Karen L Head, Bin T Teh
1Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York, USA.
Journal of Biomolecular Screening
|December 17, 2009
Summary
Optimizing gene expression analysis from formalin-fixed, paraffin-embedded tissues involves enhancing RNA recovery and reverse transcription. Using the MasterPure kit with overnight proteinase K digestion and gene-specific primers significantly improves TaqMan PCR results.
Area of Science:
- Molecular Biology
- Biotechnology
- Cancer Research
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for gene expression analysis.
- TaqMan PCR is a common method for quantifying gene expression.
Purpose of the Study:
- To compare RNA recovery methods from FFPE renal tumor tissues.
- To optimize RNA isolation and reverse transcription for improved TaqMan PCR sensitivity.
Main Methods:
- Compared five RNA isolation kits, including commercial options.
- Modified RNA isolation protocols to include overnight Proteinase K digestion.
- Evaluated two reverse transcription methods: random primers vs. gene-specific primers.
Main Results:
- The MasterPure kit yielded the highest RNA, though not significantly different from TRIzol.
- Overnight Proteinase K digestion significantly increased RNA yield.
- Gene-specific primers for reverse transcription significantly enhanced cDNA detectability by TaqMan PCR.
Conclusions:
- Optimized protocol for FFPE gene expression analysis using TaqMan qPCR involves the MasterPure kit.
- Incorporating overnight Proteinase K digestion and gene-specific primers enhances RNA yield and cDNA quantity, respectively.
