TRANSCRIPTOME 2002: From Functional Genomics to Systems
A Novel Platform for High-Sensitivity Analysis of Cell-Specific Gene Expression using Microarrays
Rajiv Raja, Jim Stanchfield, Mark Erlander and Steve Kunitake, Arcturus, Inc., Mountain View, CA
Gene expression profiles of thousands of genes can be monitored in parallel using microarrays. Microarray technology has proven to be a valuable tool in studying normal and induced variations in gene expression, thereby helping to understand the molecular mechanism of diseases, and aiding in disease diagnoses and drug discovery. However, whole tissue biopsies are typically used for these studies because microgram amounts of RNA are required for performing microarray hybridizations. Hence, measurements of molecular signals are averaged across several dissimilar cell types, resulting in a decrease in assay sensitivity. A new platform has been recently developed by Arcturus that attains improved sensitivity at 3 stages: (1) capturing pure cell populations through laser capture microdissection (LCM) while maintaining integrity of cellular RNA, (2) efficiently recovering good quality RNA from very small samples through an optimized RNA isolation system, and (3) faithfully amplifying messages from cellular RNA to provide adequate amounts of amplified antisense RNA (aRNA) for microarray analysis. First, thin tissue sections are prepared from biopsies and are fixed on microscope slides using a protocol optimized for preserving RNA quality during the process. Using laser capture microdissection (LCM), small populations of specific cell types are quickly isolated from these sections. Miniaturized devices are then used to extract and purify RNA from the captured cells in both high quality and yield. Nanogram amounts of the recovered RNA are then amplified to produce microgram quantities of aRNA using a novel linear amplification process. aRNA is then labeled and hybridized to microarrays. When the whole platform is used for gene expression analysis, considerably higher sensitivity in quantifying differential expression is attained compared to traditional approaches. Moreover, the system is capable of revealing signatures that are not seen when whole tissues are assayed. When differential expression profiles revealed by unamplified samples are compared to those from amplified samples, we see a very high correlation of r = 0.91. Labeled cDNA prepared from aRNA is routinely used in our laboratories to probe microarrays for identification of differentially expressed genes. Using this platform, molecular signatures have been identified using as few as 250 cells from frozen breast cancer biopsies, suggesting direct application to clinical chemical analyses.
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