A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards.
The quantitative measurement of gene expression requires consistent and reliable standards. At least two categories of standards, endogenous and exogenous, are currently used for quantitative PCR. The reliability of these two methods, however, has not been carefully compared. We hypothesized that a reliable quantitative PCR assay would be able to detect known dilutions of a given single-stranded (ss-) cDNA. By measuring VEGF ss-cDNA copy numbers or signal ratios of GAPDH/VEGF in 10x and 100x diluted samples of two original ss-cDNA preparations, an exogenous recombinant DNA standard (a VEGF-mimic plasmid) and an endogenously expressed GAPDH standard were tested for their ability to detect dilution factors. Using the recombinant DNA standard, the dilution factor was detected as 10.3 and 135.0 in 10x and 100x diluted samples of the original CaSki cell ss-cDNA, respectively. The detected dilution factors were 12.3 and 226.2, respectively, in 10x and 100x diluted ss-cDNA from U-251 MG cells. On the other hand, with the endogenous GAPDH standard, the dilution factors were detected as 2.7 and 8.0 in the same 10x and 100x dilutions of the original U-251 MG cell ss-cDNA. Using the same endogenous GAPDH standard, the detected dilution factors were both 4.8 in 10x and 100x dilutions of the original CaSki cell ss-cDNA. It was also found that the number of endogenous copies of GAPDH mRNA was about 1000 times higher than VEGF. The high internal lockup ratio of GAPDH vs VEGF copy numbers and the requirement for additional primer pairs make the use of an abundant endogenous standard an unreliable choice in quantitative or semi-quantitative PCR. In contrast, exogenous standard-based quantitative PCR was shown to be an accurate and reliable method for the quantitation of gene expression.
Ke
L D
LD
Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. kelidao@audumla.mdacc.tmc.edu
Chen
Z
Z
Yung
W K
WK
D016543:Central Nervous System Neoplasms / Q000235:genetics; D004277:DNA, Single-Stranded; D016228:Endothelial Growth Factors / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D005910:Glioma / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D008222:Lymphokines / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000235:genetics; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D004277:DNA, Single-Stranded; D016228:Endothelial Growth Factors; D008222:Lymphokines; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C068142:glyceraldehyde 3-phosphate dehydrogenase (304-313); D005987:Glyceraldehyde-3-Phosphate Dehydrogenases