title: Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.
abstract: BACKGROUND
  Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays.
  
  
  RESULTS
  The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases.
  
  
  CONCLUSIONS
  RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.
authors.affiliation: Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Hongyang
authors.initials: H
authors.lastname: Wang
authors.forename: James D
authors.initials: JD
authors.lastname: Owens
authors.forename: Joanna H
authors.initials: JH
authors.lastname: Shih
authors.forename: Ming-Chung
authors.initials: MC
authors.lastname: Li
authors.forename: Robert F
authors.initials: RF
authors.lastname: Bonner
authors.forename: J Frederic
authors.initials: JF
authors.lastname: Mushinski
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D007834:Lasers; D008739:Methyl Green / Q000494:pharmacology; D051379:Mice; D042282:Microdissection / Q000379:methods*; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011754:Pyronine / Q000494:pharmacology; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D012313:RNA; D008739:Methyl Green; D011754:Pyronine