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Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

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 histologica...

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Autores principales: Wang, Hongyang, Owens, James D, Shih, Joanna H, Li, Ming-Chung, Bonner, Robert F, Mushinski, J Frederic
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1513394/
https://www.ncbi.nlm.nih.gov/pubmed/16643667
http://dx.doi.org/10.1186/1471-2164-7-97
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author Wang, Hongyang
Owens, James D
Shih, Joanna H
Li, Ming-Chung
Bonner, Robert F
Mushinski, J Frederic
author_facet Wang, Hongyang
Owens, James D
Shih, Joanna H
Li, Ming-Chung
Bonner, Robert F
Mushinski, J Frederic
author_sort Wang, Hongyang
collection PubMed
description 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. CONCLUSION: 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.
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spelling pubmed-15133942006-07-21 Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA Wang, Hongyang Owens, James D Shih, Joanna H Li, Ming-Chung Bonner, Robert F Mushinski, J Frederic BMC Genomics Research Article 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. CONCLUSION: 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. BioMed Central 2006-04-27 /pmc/articles/PMC1513394/ /pubmed/16643667 http://dx.doi.org/10.1186/1471-2164-7-97 Text en Copyright © 2006 Wang et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Wang, Hongyang
Owens, James D
Shih, Joanna H
Li, Ming-Chung
Bonner, Robert F
Mushinski, J Frederic
Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA
title Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA
title_full Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA
title_fullStr Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA
title_full_unstemmed Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA
title_short Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA
title_sort histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular rna
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1513394/
https://www.ncbi.nlm.nih.gov/pubmed/16643667
http://dx.doi.org/10.1186/1471-2164-7-97
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