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Chronic Fatigue Syndrome > Publications > Molecular Epidemology Publications >

Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.

Rajeevan MS, Ranamukhaarachchi DG, Vernon SD, Unger ER.
Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies.
Methods 25:443-451, 2001.

Summary

The CDC CFS Molecular Epidemiology Program is using microarrays and differential display-polymerase chain reaction (DD-PCR) to search for gene expression patterns that identify persons with CFS. These extremely powerful technologies have identified prognostic markers for lymphomas, prostate, and breast cancers but have not been applied to studies of diseases such as CFS. Both methods have inherent limitations in reliability and genes identified as differentially expressed must be validated with another method. This report provides a detailed description of the real-time polymerase chain reaction methodology and its integration with DNA array and DD-PCR technologies for validation of differentially expressed genes.

Abstract

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies. Since SYBR Green I dye is a nonspecific intercalating dye, the reaction is made specific by using "hot-start" PCR and empirically determined annealing and signal acquisition temperatures for each gene-specific primer. Relative expression levels were quantified by constructing a standard curve using cDNA dilutions of a highly expressed gene. Using this approach, real-time PCR validated 17 of 21 (71%) genes identified by DNA arrays, and all but 1 or 13 (91%) genes identified by differential display PCR (DD-PCR). Validation of differentially expressed genes detected by array analysis was related to hybridization intensity. Real-time RT-PCR results suggest that genes identified by DNA arrays with a two or fourfold difference in expression cannot be accepted as true or false without validation. Validation of differentially expressed genes detected by DD-PCR was not affected by band intensities. Regardless of the gene expression profiling technology (microarrays, DD-PCR, serial analysis of gene expression and subtraction hybridization), once the sequence of the gene of interest is known, the real time RT-PCR approach is well suited for validation of differential expression since it is quantitative and rapid and requires 1000-fold less RNA than conventional assays.

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