Evaluation and application of RNAs derived from laser microdissected specimens using DNA microarray for expression genomics

Laser capture rnicrodissection (LCM) technique has been widely applied to isolate a homogeneous population of cells from heterogeneous tissues. Combined use of microdissection and large-scale DNA microarrays is a powerful tool to research global transcriptional landscape of the clinical specimens. However, the scope of performing microarray using intact RNA derived from LCM isolated cells is very limited due to the instability of mRNA during the LCM process. To overcome this issue, we estimated limitation of the amount of RNA and optimized the RNA extraction procedure for LCM-derived RNA to reproduce RNA with consistent quality for DNA chip application. The three different RNA extraction methods, and amplification methods for LCM-derived RNAs were compared for better quality of hybridization performance of DNA chips. The Trizol reagent method produced RNA of better quality and yield in the LCM-derived samples. However, amplification of LCM-derived RNA did not fully compensate for the RNA content of amplified RNA (cRNA) derived from the direct dissection of tissue samples. In addition, hybridization performance of LCM-derived cRNAs was not better than that of direct dissection-derived cRNAs, without using LCM. For the reproducibility of data, the correlation coefficients of the samples derived from the two methods were similar, but reproducibility between the two methods was not highly significant (R=0.762). Taken together, our results suggest that application of LCM-derived RNAs to DNA microarrays may be limited in the quantity or quality of cRNA, and may generate less hybridization potential in DNA microarray experiments, but this laser-based microdissection method can be applied to conduct more precise and exquisite assessment of gene expression analysis in heterogeneous tissue samples.