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.

Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity.

UNLABELLED: Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.