Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.
Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.
Su
Jack Meng Fen
JM
Laboratory of Molecular Neuro-oncology, Cancer Genomics Program, Texas Children's Cancer Center, Houston, Tex., USA.
Perlaky
Laszlo
L
Li
Xiao-Nan
XN
Leung
Hon-Chiu Eastwood
HC
Antalffy
Barbara
B
Armstrong
Dawna
D
Lau
Ching C
CC
D000818:Animals; D001921:Brain / Q000033:anatomy & histology*; D000431:Ethanol; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods; D007834:Lasers; D051379:Mice; D042282:Microdissection; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods
D012333:RNA, Messenger; D005557:Formaldehyde; D000431:Ethanol