Improved RNA quality and TaqMan super( registered )Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

• Published in: BMC biotechnology 2008-01, Vol.8, p.10-10
• Main Authors: Li, Jinghuan, Smyth, Paul, Cahill, Susanne, Denning, Karen, Flavin, Richard, Aherne, Sinead, Pirotta, Marco, Guenther, Simone M, O'Leary, John J, Sheils, Orla
• Format: Article
• Language: English
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-8-10

Background Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62-164 bp). Results Results from the Bioanalyzer and TaqMan super( registered )data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70 degree C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan super( registered )detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan super( registered )PreAmp consistently achieved decreased C sub(T )values in both snap frozen and FFPE aliquots compared with no pre-amplification. Conclusion Modification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan super( registered )PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.