Primary Navigation for the CDC Website
CDC en Español

Search:  

News & Highlights

Chronic Fatigue Syndrome > Publications > Molecular Epidemology Publications >

Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis.

Rajeevan MS, Dimulescu IR, Vernon SD, Verma M, Unger ER
Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis
Genomics 2003; 82:491-497.

Summary

High-throughput gene expression profiling methods have allowed biologists to take a broad and open approach to the discovery of novel diagnostic markers. Each potential disease marker requires careful validation in well-designed epidemiologic studies to test the strength of the association or predictive value. However, the extremely limited amount of total RNA (often less than 1 - 5 micrograms derived from 5-20 million whole blood cells available in a CFS sample) and the lability of RNA during long-term storage greatly limit the numbers of studies that these collections can support. We are exploring methods for long-term storage of samples during the extended period of biomarker discovery and validation.

Our goal was to develop a method that would make RNA in biorepositories very close to a stable renewable resource that could be directly used in all approaches to gene expression profiling (microarray, DD-PCR, RT-PCR, RNAse protection assays). Key features of the procedure include: 1) a modified poly-dT primer to direct reverse transcriptase to polyA transcripts and to incorporate anchoring and restriction enzyme (cloning) sites; 2) use of the template switching activity of reverse transcriptase to incorporate a second primer with anchoring, restriction enzyme (cloning) and RNA polymerase binding sequences at the 3' end of the single-stranded first cDNA product; and 3) use of anchor-primed limited PCR to globally copy all products to double-stranded cDNA products for archiving. When needed, the archived material is used as template for in vitro transcription (IVT) from the upstream RNA polymerase site to yield amplified RNA in the sense direction (sRNA).

The size of sRNA we synthesized ranged from 3.0- 0.1 kb. Real-time RT-PCR estimated 2000-2500-fold amplification of glyceraldyde-3-phosphate dehydrogenase in Caski total RNA by sRNA synthesis. Real-time RT-PCR also determined that sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging 5 orders of magnitude (0.00001-0.1%). This reflects the high-fidelity of sRNA synthesis for mRNAs as low as 0.3-30 copies/cell. sRNA synthesis was successful with RNA from cell lines and human tissues. The double-stranded cDNA is engineered to construct cell-type specific libraries and as a stable, renewable archive of original RNA.

Abstract

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA. Limited PCR was used for double-stranded DNA synthesis. sRNA was synthesized from PCR products by in vitro transcription. sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 25000-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5'-> 3' direction, the appropriate template for any gene expression analysis.

Page last modified on May 8, 2006

Topic Contents

• Topic Contents

Additional Navigation for the CDC Website

“Safer Healthier People”
 Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30333, USA
Tel: 404-639-3311  /  Public Inquiries: (404) 639-3534  /  (800) 311-3435