title: Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.
abstract: 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.
authors.affiliation: Laboratory of Molecular Neuro-oncology, Cancer Genomics Program, Texas Children's Cancer Center, Houston, Tex., USA.
authors.forename: Jack Meng Fen
authors.initials: JM
authors.lastname: Su
authors.forename: Laszlo
authors.initials: L
authors.lastname: Perlaky
authors.forename: Xiao-Nan
authors.initials: XN
authors.lastname: Li
authors.forename: Hon-Chiu Eastwood
authors.initials: HC
authors.lastname: Leung
authors.forename: Barbara
authors.initials: B
authors.lastname: Antalffy
authors.forename: Dawna
authors.initials: D
authors.lastname: Armstrong
authors.forename: Ching C
authors.initials: CC
authors.lastname: Lau
mesh_terms: 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
chemical_list: D012333:RNA, Messenger; D005557:Formaldehyde; D000431:Ethanol