The cDNA microarray hybridization method is now widely used to analyze simultaneously the expression of thousands of genes in cancer tissues [5
], but the contamination of tumors by epithelial, stromal or immune cells presents problems for obtaining accurate expression profiles. Microarray analysis coupled with LCM is a good way to solve this problem. However, cDNA microarray analysis requires a large amount of total RNA (5 μg to 100 μg). It has been shown that one or two rounds of amplification can be used reliably, without compromising RNA quality or gravely skewing patterns of gene expression, when only small initial amounts of total RNA are available [16
]. In this study, we used two rounds of amplification to obtain amounts of antisense RNA sufficient for microarray hybridization yielding strong signal intensities, permitting statistical data processing.
To positively identify the specific cells desired for the microdissection, a staining protocol must be used, which permits identification of plasma cells in desiccated cryosections. Such sections, without cover slips, are required for microdissection and may pose a critical problem, since desiccate cryosections are not optically ideal for histological examination (cf. Figure ). However, staining solutions may react with cellular components, such as RNA, potentially adversely affecting RNA integrity. In addition, the aqueous components of the staining reagents may contain or activate intracellular RNAases and result in RNA degradation. For this reason, it was important to test different conditions for tissue staining and section dehydration to assure high quality of RNA from LCM samples. To assure ourselves of adequate numbers of uniform populations of plasma cells, which are scarce in most tissues, we utilized pellets of cultured plasma cell tumor cells that were sectioned and stained as if they had been collected by LCM. We used this approach, because obtaining large enough sample of plasma cell tumor cells from tissues by LCM would have been impracticable.
Our modification (addition of RNAase inhibitor) to the standard MGP staining method has made it suitable for use with LCM. Sections treated as described here can be used to prepare total RNA of good quality. Though pyronin is a fluorescent molecule that can bind to RNA, we detected only 21 changes when compared with unstained LCM samples. This amounted to less than 0.3% of the analyzed microarray elements, and less than one half of the number changed due to the other staining methods tested.
It appears that inclusion of RNAase inhibitor in staining solutions can prevent RNA from damage by endogenous and exogenous RNAase. The Nissl-stained group was the only staining protocol that did not include RNAase inhibitor. The number of genes in this group with 2-fold changes, 70 spots, was the highest among all four groups, indicating that this staining procedure damaged the most mRNAs.
In summary, we performed a statistical analysis of 16 cDNA microarray chips comparing fluorescent-labeled cDNA generated from amplified RNA from 16 independently isolated and processed, LCM-procured, stained frozen sections with amplified RNA from an identical pellet of plasma cell tumor cells. This showed that a variable amount of spot fall-out was seen after staining of the frozen sections, depending on the staining method, indicating that the choice of staining method can be important in minimizing loss of mRNAs from microarray analysis of LCM-derived RNA. Happily, the degree of mRNA damage was small with the commonly used H&E method, so long as RNAase inhibitor was included in the aqueous hematoxylin solution. Nissl staining solution, which may not be compatible with RNAase inhibitor addition, introduced the greatest degree of damage. Fortunately, for our ongoing study of plasma cell tumor development, the MGP stain, chosen for its unique ability to flag plasma cells in tissue sections, was the least destructive of all. Our analysis suggests that maximizing the use of non-aqueous staining solutions is recommended for other specialty stains that might be advantageous for identification of other cell types in studies that require use of LCM. The inclusion of RNAase inhibitor in aqueous solutions appears to be important in rendering histological staining procedures suitable to extraction of intact RNA.