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.

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.

Figure 1

Histochemical staining of frozen sections of an intraperitoneal primary mouse plasma cell tumor. A nodule rich in plasma cells can be seen in the midst of surrounding stromal and fat cells. The stains of the sections are as follows: H&E, hematoxylin and eosin; Nissl; MGP (methyl green pyronin) and kappa (immunoperoxidase in the top four panels or immunofluorescence in the bottom four panels) staining of immunoglobulin kappa light chains). The images in the top four panels represent frozen sections stained and mounted under cover slips in a distant histology laboratory and captured using high quality optics in a photographic studio. Those in the bottom four panels reflect the challenges of interpreting microscopic views on a microscope optimized for LCM, viewing frozen sections that had been processed and stained with the utmost speed to minimize RNA damage and lacking cover slips. Ovals indicate clusters of plasma cells with characteristic MGP staining and rich in Ig κ protein.

Figure 2

Analysis of RNA quality. One ng of total RNA from one sample from each staining group was electrophoresed on Agilent Bioanalyzer system (Pico RNA chip). RNA ladder 6000 (Ambion, Inc). Lane A contains total RNA from unsectioned cell pellets. Lanes marked MGP, H&E, NS and IF indicate the staining method used before LCM procurement and RNA preparation. Lowermost band (green) in all lanes is an Agilent alignment marker.

Figure 3

Log-ratios of genes significantly different between A (cell pellet) and LCM samples where genes are sorted according to the order of a dendrogram generated by clustering over genes. Red shades correspond to higher expression in the staining group than A; green shades correspond to lower expression; and black indicates no difference.