A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients.
OBJECTIVE
Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients.


METHODS
All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors.


RESULTS
The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively.


CONCLUSIONS
This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.
Chang
Joseph T
JT
Department of Radiation Oncology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
Chen
I-How
IH
Liao
Chun-Ta
CT
Wang
Hung-Ming
HM
Hsu
Yuan-Ming
YM
Hung
Kuao-Fu
KF
Lin
Chia-Ju
CJ
Hsieh
Ling-Ling
LL
Cheng
Ann-Joy
AJ
D016228:Endothelial Growth Factors / Q000235:genetics* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008222:Lymphokines / Q000235:genetics* / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D010982:Platelet-Derived Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2