Improvement of tissue preparation for laser capture microdissection: Application for cell type-specific miRNA expression profiling in colorectal tumors

Abstract

Background: Laser capture microdissection (LCM) has successfully isolated pure cell populations from tissue sections and the combination of LCM with standard genomic and proteomic methods has revolutionized molecular analysis of complex tissue. However, the quantity and quality of material recovered after LCM is often still limited for analysis by using whole genomic and proteomic approaches. To procure high quality and quantity of RNA after LCM, we optimized the procedures on tissue preparations and applied the approach for cell type-specific miRNA expression profiling in colorectal tumors.Results: We found that the ethanol fixation of tissue sections for 2 hours had the maximum improvement of RNA quality (1.8 fold, p = 0.0014) and quantity (1.5 fold, p = 0.066). Overall, the quality (RNA integrity number, RIN) for the microdissected colorectal tissues was 5.2 ± 1.5 (average ± SD) for normal (n = 43), 5.7 ± 1.1 for adenomas (n = 14) and 7.2 ± 1.2 for carcinomas (n = 44). We then compared miRNA expression profiles of 18 colorectal tissues (6 normal, 6 adenomas and 6 carcinomas) between LCM selected epithelial cells versus stromal cells using Agilent miRNA microarrays. We identified 51 differentially expressed miRNAs (p