Transcriptional profiling of tissue biopsies using microarrays has become a powerful tool for studying biological mechanisms and their alterations leading to diseases. Laser capture microdissection (LCM) has proven its value as a technique for the isolation of desired cell populations from tissue biopsies, thereby improving the sensitivity of microarray analysis. Historically, the ability to utilize pure cell populations obtained through LCM for microarray analysis has been severely limited by the need for several micrograms of total RNA to perform hybridizations. The system for Microgenomics® provides researchers with a complete and robust solution for microarray analysis from small samples, including systems for (a) microdissecting pure cell populations from tissue biopsies, (b) isolating high quality RNA from microscopic samples, (c) amplifying picogram amounts of total RNA isolated from frozen tissue, (d) non-enzymatic labeling of amplified antisense RNA (aRNA) for microarray hybridizations, and (e) scanning arrays and analyzing microarray data. This integrated platform includes LCM instrumentation to capture specific cells of interest using a nondamaging near-infrared (IR) laser and incorporates protocols and procedures to analyze the quality of samples at various stages during the process. The microgenomics platform ensures high- efficiency recovery of quality total RNA from as little as a single cell obtained through LCM. High-sensitivity linear amplification of mRNA is possible from as little as 100 picograms (10 LCM cells) of total RNA, generating enough aRNA for replicate microarray hybridizations. Linear amplification is followed by non-enzymatic labeling, which allows the use of unlabeled nucleotides during amplification. This significantly reduces the required amount of starting material and results in higher aRNA yields and higher %P calls during microarray analysis. The complete microgenomics platform has been validated for use with all common microarray platforms.
In this application note gene expression profiling of human ovarian cancer and human breast cancer cells captured using LCM and laser-cutting (LC) on the LCM microdissection instrument. Subsequent to LCM, RNA was extracted, linearly amplified and labeled for microarray hybridization and analysis.