A method for the absolute quantification of cDNA using real-time PCR.
Real-time PCR is an extremely powerful technique, however, often its results are open to interpretation since there is no convention for data presentation. This anomaly has arisen because many applications rely on non-standard calibration genes, which themselves often change in value during experimental manipulation. We present a novel method for absolute quantification of cDNA species using a combination of extremely accurate double-stranded DNA quantification and a plasmid reference curve. PicoGreen and reference standards are used to measure the amount of cDNA present in a sample using fluorescence. Real-time PCR products are cloned into plasmids and then used to calibrate unknown samples. This cloning is achieved using the same primers necessary for real-time PCR and thus does not involve a second design stage. Results are expressed as copy number per microgram of oligo-dT primed cDNA and consequently may be compared between both inter and intra-experimentally. We show results from a sample human system in which absolute levels of interferon-gamma, TNF-alpha, interleukin-2 and interleukin-10 are measured. We further compare the copy numbers of these genes with levels of released protein and find remarkable correlation. Although our interest has been cytokine quantification, we believe that this technique is widely applicable to the majority of real-time PCR applications.
Whelan
Joseph A
JA
Onyvax Ltd., St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
Russell
Nick B
NB
Whelan
Michael A
MA
D016207:Cytokines / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis*; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D018189:Immunomagnetic Separation; D008214:Lymphocytes / Q000502:physiology; D009930:Organic Chemicals; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D012333:RNA, Messenger