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Open Access Highly Accessed Research article

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

Hongyang Wang15, James D Owens1, Joanna H Shih2, Ming-Chung Li3, Robert F Bonner4 and J Frederic Mushinski16*

Author Affiliations

1 Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA

2 Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, MD 20892, USA

3 The Emmes Corporation, Rockville, MD 20850, USA

4 Laboratory of Integrative and Medical Biophysics, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA

5 Division of Rheumatology and Immunology, Department of Internal Medicine, University of Virginia, Charlottesville, VA 22906, USA

6 Bldg. 37, Room 3134C, NIH, 37 Convent Drive, MSC-4258, Bethesda, MD 20892-4258, USA

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BMC Genomics 2006, 7:97  doi:10.1186/1471-2164-7-97

Published: 27 April 2006

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.

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.