Comparison of different standards for real-time PCR-based absolute quantification.
Quantitative 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 (R(2))) using hydrolysis probe (TaqMan) chemistry. Results, expressed as copy numbers/microl, are presented from a sample human system in which absolute levels of HuPO (reference gene) and the cytokine gene IFN-gamma were measured. To ensure the suitability and stability of the four standards, the experiments were performed at 0, 7 and 14 day intervals and repeated 6 times. We have found that the copy numbers vary (due to degradation of standards) over the period of time during storage at 4 degrees C and -20 degrees C, which affected PCR efficiency significantly. The cloned target sequences were noticeably more stable than the PCR product, which could lead to substantial variance in results using standards constructed by different routes. Standard quality and stability should be routinely tested for assays using qPCR.
Dhanasekaran
S
S
Infectious Diseases Unit, St. John's Research Institute, Koramangala, Bangalore-560034, India.
Doherty
T Mark
TM
Kenneth
John
J
D003001:Cloning, Molecular; D017931:DNA Primers / Q000592:standards*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D011786:Quality Control; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D012269:Ribosomal Proteins / Q000235:genetics*; D013997:Time Factors
D017931:DNA Primers; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D007371:Interferon-gamma