A method for the absolute quantification of cDNA using real-time PCR

Onyvax Ltd., St. George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK Received 3 February 2003; received in revised form 1 May 2003; accepted 1 May 2003

Abstract 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 doublestranded 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-g, TNF-a, 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.D 2003 Elsevier B.V. All rights reserved. Keywords: Absolute quantification; Real-time PCR; Cytokines

1. Introduction Conventional reverse-transcription polymerase chain reaction (RT-PCR) has often been applied to the detection of relatively rare mRNA transcripts, however, it is not truly quantitative and is often a gross underestimate of total message levels actually present, mostly due to depletion of reagents during the reaction (Santagati et al., 1997). Consequently, when PCR products are visualised on a gel, the intensity of the bands are not proportional to the amount of initial target, thus making image analysis highly dubious.

One of the most accurate methods to overcome this limitation is to use the 5V–3Vexonuclease reaction as exemplified by the ABI Prism 7700 sequence detector (Holland et al., 1991). In such real-time PCR, a probe lies downstream of the forward primer and has a fluorescent tag at the 5Vend and a quencher at the 3Vend. As the PCR proceeds, Taq polymerase uses its 5V–3Vexonuclease activity and destroys the probe thus generating fluorescence, which is proportional to the amount of amplified target DNA present. Hence, if fluorescence is rapidly detected, then large amounts of DNA are present and vice versa when release is slow.