Abstract
BackgroundArchival 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).ResultsResults from the Bioanalyzer and TaqMan® data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70°C for 20 minutes was determined to be the best condition of those tested to disrupt cross-links while not compromising RNA integrity. TaqMan® detection was influenced by master mix, amplicon size and the incorporation of a pre-amplification step. TaqMan® PreAmp consistently achieved decreased CT values in both snap frozen and FFPE aliquots compared with no pre-amplification.ConclusionModification to extraction protocols has facilitated procurement of RNA that may be successfully amplified using QRT-PCR. TaqMan® PreAmp system is a robust and practical solution to limited quantities of RNA from FFPE extracts.
Highlights
Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing
RNA extraction RNA quantity was assessed spectrophotometrically using NanoDrop® ND-1000 Spectrophotometer (Wilmington, USA), which showed that the yields from snap frozen extracts were greater than those from FFPE when RNA was extracted from identical numbers of cells using all the protocols examined (Stratagene Absolutely RNA® FFPE Kit, Ambion RecoverAllTM Total Nucleic Acid Isolation Kit, Gentra Purescript® RNA Purification Kit and Invitrogen Trizol® Reagent)
Normal thyroid cell lines were split into two aliquots: One was snap frozen, the other formalin fixed and paraffin embedded
Summary
Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; 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 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). BMC Biotechnology 2008, 8:10 http://www.biomedcentral.com/1472-6750/8/10 nosis and prognosis, and potentially elucidate novel therapeutic targets [1,2] These tissues have not been widely used in molecular biology due to the degradation and chemical modification of RNA extracted from FFPE blocks. The situation is made more complicated by the fact that RNA is modified by methylol groups to form cross-links with protein or nucleic acid during formalin fixation [4,5,6], which results in poor yields [1,7] and compromised extracts
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