qPCR absolute quantification.pdf

Journal of Immunological Methods 354 (2010) 34–39 Contents lists available at ScienceDirect Journal of Immunological Methods j ourna l homepage: www.e lsev ie / locate / j imResearch paper Comparison of different standards for real-time PCR-based absolute quantification S. Dhanasekaran a,b, T. Mark Doherty c,d, John Kenneth a,? and TB Trials Study Group a Infectious Diseases Unit, St. Johns Research Institute, Koramangala, Bangalore – 560 034, India b The Gade Institute, Section for Microbiology and Immunology, University of Bergen and Haukeland University hospital, N-5021, Norway c Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, Copenhagen 2300s, Denmark d Center for International Health, University of Bergen, N-5021, Norwaya r t i c l e i n f oAbbreviations: qPCR, Quantitative real-time PCR; R2, Correlation coefficient; HuPO, Human acidic ribos IFN γ, Interferon gamma. ? Corresponding author. Tel.: +91 80 fa E-mail address: johnkennt@ (J. Kenneth 0022-1759/$ – see front matter ? 2010 Elsevier B.V. doi:10.1016/j.jim.2010.01.004a b s t r a c tArticle history: Received 3 October 2009 Received in revised form 8 January 2010 Accepted 8 January 2010 Available online 25 January 2010Quantitative real-time PCR (qPCR) is a powerful tool used for both research and diagnostic, which has the advantage, compared to relative quantification, of providing an absolute copy number for a particular target. However, reliable standards are essential for qPCR. In this study, we have compared four types of commonly-used standards — PCR products (with and without purification) and cloned target sequences (circular and linear plasmid) for their stability during storage (using percentage of variance in copy numbers, PCR efficiency and regression curve correlation coefficient (R2)) using hydrolysis probe (TaqMan) chemistry. Results, expressed as copy numbers/μl, are presented from a sample human system in which absolute levels of HuPO (reference gene) and th