Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR.
BACKGROUND
Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as beta-actin (ACTB).


RESULTS
The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns.


CONCLUSIONS
StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further, it correlates well with Taqman real time PCR in terms of quantitative and discriminatory ability. This label-free, inexpensive technique may provide the ability to generate prognostically important molecular signatures unique to individual tumors and may enable identification of novel therapeutic targets.
Pagliarulo
Vincenzo
V
Department of Pathology, University of Southern California, Keck School of Medicine, 2011 Zonal Ave, HMR 312C, Los Angeles, CA 90033, USA. vpagliarulo@urologia.uniba.it
George
Ben
B
Beil
Stephen J
SJ
Groshen
Susan
S
Laird
Peter W
PW
Cai
Jie
J
Willey
James
J
Cote
Richard J
RJ
Datar
Ram H
RH
D001667:Binding, Competitive; D002295:Carcinoma, Transitional Cell / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D003199:Computer Systems; D015342:DNA Probes / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D052138:Genes, Neoplasm / Q000235:genetics; D019942:Genes, p16; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000592:standards; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000235:genetics
D018797:Cell Cycle Proteins; D015342:DNA Probes; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D012334:RNA, Neoplasm; D014157:Transcription Factors