Immunohistochemical Validation of Expression Microarray Results

Abstract

Expression microarrays, which determine the level of expression of tens of thousands of mRNAs by a specific cell or tissue type, are powerful and increasingly more widely used investigative, diagnostic, and prognostic molecular biological tools.1,2 However, there are technical aspects to using expression microarrays that can produce results erroneously representing either under- or overexpression of specific genes. Chuaqui et al3 and Simon et al4 have discussed some of these pitfalls. For example, false negativity can result from low expression levels, transcript drop-out (attributable to inefficient priming of specific mRNA(s)), poor adhesion of DNA to the slide, and splice variants with sequences not included on the array. Conversely, sources of false positivity include repetitive nucleotide elements, poly(A) tails, and sequence homology between functionally different transcripts, an inappropriately chosen reference standard, and high background levels due to nonspecific binding of nucleotides to the microarray slides. Ways to minimize the sources of error continue to be developed. For example, use of multiple different sequences of a given gene provides a way not to under-represent a given gene. Conversely, more rigorous attention to minimizing sources of background binding of detection nucleotides can minimize over-representation of highly expressed genes.