Preservation of gene expression ratios among multiple complex cDNAs after PCR amplification: application to differential gene expression studies.
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.
Ji
W
W
Department of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. wji@genomicfx.com
Cai
L
L
Wright
M B
MB
Walker
G
G
Salgam
P
P
Vater
A
A
Lindpaintner
K
K
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D005821:Genetic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D011506:Proteins; D012333:RNA, Messenger