Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.
Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.
Yang
Wen
W
Genospectra, Inc., Fremont, CA 94555, USA. wyang@genospectra.com
Maqsodi
Botoul
B
Ma
Yunqing
Y
Bui
Son
S
Crawford
Kimberly L
KL
McMaster
Gary K
GK
Witney
Frank
F
Luo
Yuling
Y
D004247:DNA / Q000187:drug effects*; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008168:Lung / Q000737:chemistry; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D016612:Paraffin Embedding / Q000379:methods; D012333:RNA, Messenger / Q000302:isolation & purification*; D016707:Tissue Fixation / Q000379:methods
D005404:Fixatives; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D005557:Formaldehyde; D004247:DNA