Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.
Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.
D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000592:standards; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D051379:Mice; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity