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Impact of RNA extraction from limited samples on microarray results

Ojaniemi H, Evengard B, Lee DR, Unger ER, Vernon SD
Impact of RNA extraction from limited samples on microarray results
BioTechniques 2003;35:968-975.

Summary

A major goal of the CDC CFS Research Program is to identify diagnostic markers of CFS and to identify its central biochemical pathways. Our major effort has involved gene expression analysis and we continue to refine assay methodology as applied to population-based epidemiologic studies.

High throughput gene expression profiling methods have allowed biologists to take a broad and open approach to the discovery of novel diagnostic markers and molecular targets for therapeutic or preventive drug design. Each "discovered" disease marker requires careful validation in well-designed epidemiologic studies to test the strength of the association or predictive value. However, the extremely limited and finite amount of total RNA (often less than 1 - 5 micrograms from 5-20 million blood cells) available from each CFS sample and the liability of RNA during long-term storage significantly limit the numbers of studies that these priceless collections can support. We are exploring methods for archiving RNA.

This paper describes optimization of RNA extraction prior to measuring gene expression profiles.

Abstract

Impact of RNA extraction from limited samples on microarray results: To move microarray technology into the diagnostic realm, the impact of technical parameters, such as sample preparation and RNA extraction, needs to be understood and minimized. We evaluated the impact of two RNA extraction methods, DNase treatment and the amount of hybridized cDNA probe, on the outcome of microarray results. The results for both RNA extraction methods were comparable, although one method resulted in residual DNA that slightly affected the microarray results. As little as one microgram of total RNA could be used to synthesize a cDNA probe and resulted in a gene expression profile that was similar to one produced using 5 mg total RNA, even though the overall signal intensity was lower. These experiments illustrate that microarray technology holds great promise for the use of limited clinical samples in the diagnostic setting.

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