Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials.
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 data showed improvement of RNA quality extracted using the modified protocols from FFPE. Incubation at 70 degrees 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.


CONCLUSIONS
Modification 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.
Li
Jinghuan
J
Deptment of Histopathology, University of Dublin, Trinity College, Dublin, Ireland. jinghual@tcd.ie
Smyth
Paul
P
Cahill
Susanne
S
Denning
Karen
K
Flavin
Richard
R
Aherne
Sinead
S
Pirotta
Marco
M
Guenther
Simone M
SM
O'Leary
John J
JJ
Sheils
Orla
O
D005404:Fixatives; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D016612:Paraffin Embedding / Q000379:methods*; D011786:Quality Control; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
D005404:Fixatives; D005557:Formaldehyde; D012313:RNA