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Chronic Fatigue Syndrome > Publications > Molecular Epidemology Publications >

Reproducibility of alternate probe synthesis approached for gene expression profiling with arrays.

Vernon SD, Unger ER, Rajeevan M, Dimulescu IM, Nisenbaum R, Campbell CE.
Reproducibility of alternate probe synthesis approached for gene expression profiling with arrays.
J Mol Diag 2:124-127, 2000.

Summary

This is the second paper published from CDC's CFS Molecular Epidemiology Program. We performed this study to determine the best method for labeling small amounts of RNA that could be used in studies of gene expression profiling. We demonstrated that a method of labeling total RNA that includes an amplification step (RT-PCR labeling with the SMARTT system) gave reproducible results that were representative of the original sample even when a very small amount of sample was used (1µg).

Abstract

Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high density filter arrays (HDFA) are synthesized from 1 Fg of purified poly (A)+ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to HDFAs, 1) reverse transcription (RT) of 5 Fg total RNA and 2) RT-PCR of 1 Fg total RNA using the SMARTT system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMARTT cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA and 122 (81%) by the SMARTT cDNA probe. We conclude that SMARTT cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard.

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