Welcome to the CDC Chronic Fatigue Syndrome site.
Skip directly to the search box, site navigation, or content.
News & Highlights
- CFS Public Health Research Program 5-year Strategic Plan *NEW*
CFS Research Program, External Peer Review (PDF – 46KB)- New Publication: 1994 CFS Case Definition Criteria Validated
- New Publication Linking Sexual Abuse and CFS
- Current CDC Research
- CME Course
- Chronic Fatigue Registry
- Treatment Guidelines
- Other Federal Resources
- Brochures
- Glossary
- more...
- Mission/Goals
- Public Service Announcements
- Brochures
- Photo Exhibit
CFS Awareness
Campaign
Call to Action - Act Now
| Paid for by | |
| HHS | CDC |
 |
 |
Modifying differential display polymerase chain reaction to detect relative changes in gene expression profiles.
Ranamukhaarachchi DG, Rajeevan MS, Vernon SD, Unger ER.Modifying differential display polymerase chain reaction to detect relative changes in gene expression profiles.
Anal Biochem 306:343-346, 2002.
Summary
Differential display-polymerase chain reaction (DD-PCR) makes a DNA copy (cDNA) of messenger RNA (mRNA) and reproduces (amplifies) this cDNA many times by means of multiple separate polymerase chain reactions (PCR). DD-PCR analysis of mRNA results in complete coverage and detection of all known and unknown expressed genes in a sample and can be used to identify cellular and viral biomarkers on the basis of alterations in gene expression between samples from diseased (CFS) and not diseased (controls) persons. DD-PCR has the advantage of discovering novel genes.
Although DD-PCR is robust and reproducible, the procedure required optimization in terms of its 1) ability to detect the common differences in gene expression levels (2-10 fold), 2) suitability with small amounts of RNA from peripheral blood cells (PBMC), 3) suitability with fluorescence detection, and 4) validation by real-time RT-PCR. This is the program's first publication describing optimization of a fluorescent DD-PCR procedure for genome-wide expression profiling with as little as 1 microgram total RNA.
Abstract
DD-PCR is an increasingly important tool for gene expression profiling studies. Since the introduction of DD-PCR, numerous advances have been made in minimizing false positives and in adapting the technology to multicolor fluorescence detection. However, DD-PCR band intensities do not accurately reflect expression levels since 100-fold differences in the amount of input RNA give comparable results. Therefore, current DD-PCR conditions would fail to detect differentially expressed genes within the 10-fold range that microarray studies have identified as the most common level of regulation.
In this study, we evaluate the impact of changing the amount of input cDNA and the number of high-stringency PCR cycles on the ability of fluorescent DD-PCR to produce complex expression profiles with band intensities that reflect known 2- to 10-fold dilutions of target. We found that for most primer combinations, 4-fold less cDNA and high-stringency PCR cycles reduced to 25 produced reproducible complex band patterns with intensities that reflected 2- to 10-fold differences in expression levels. Real-time quantitative PCR confirmed 90% of differentially expressed genes detected by this modified DD-PCR. These simple changes can be used to make fluorescence DD-PCR more quantitative and may be applied to related differential display techniques, such as RNA arbitrarily primed PCR (RAP-PCR).
Page last modified on May 8, 2006
Additional Navigation for the CDC Website
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