Preservation of Gene Expression Ratios Among Multiple Complex cDNAs After PCR Amplification: Application to Differential Gene Expression Studies |
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Authors: | Ji Wan Cai Li Wright Matthew B Walker Gaby Salgam Prathima Vater Axel Lindpaintner Klaus |
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Institution: | (1) Department of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA;(2) Roche Genetics, F. Hoffmann-La Roche, CH-4070 Basel, Switzerland;(3) Department of Vascular and Metabolic Diseases, F. Hoffmann-La Roche, CH-4070 Basel, Switzerland |
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Abstract: | Comparative gene expression studies are often limited by low availability of tissue and poor quality of extractable mRNA.
Collective PCR amplification of minute quantities of mRNA has great potential for overcoming these limitations. However, there
remains significant concern about the effects of amplification on the absolute and relative abundance of individual mRNAs
that could complicate subsequent gene expression studies. To address this problem, we systematically compared the relative
abundance of many specific mRNAs from complex cDNA preparations (from tissue and cultured cells) both before and after amplification
by PCR. Our results demonstrated that, as expected, the absolute abundance of different mRNAs in a cDNA library is altered
in an unpredictable manner by PCR amplification. However, we found that the concentration ratios of specific mRNAs among different
cDNA preparations were routinely well conserved after PCR amplification. Thus, for the purpose of comparative expression studies
for specific mRNAs in two (or more) complex cDNAs, PCR-amplified cDNA is equally useful as unamplified cDNA. These results
provide a rigorous experimental validation and offer a theoretical treatment to support the utility of PCR amplified cDNA
for differential gene expression studies. We conclude that the inherent difficulties in performing differential screening
studies such as gene chip and array analyses on limited amounts of biological materials can be overcome by a PCR amplification
step without compromising data quality.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | cDNA PCR cDNA TaqMan Analysis gene expression Pearson's correlation |
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