[A] The QuantiNova Probe PCR Kit was used to detect the single-copy gene, CFTR, in leukocyte genomic DNA using 30, 3, 0.3, and 0.03 ng gDNA, corresponding to 104 to 10 copies, and the results were plotted to create a calibration curve. The plot of copy number versus CT value demonstrates high linearity. A master mix sufficient for 60 reactions was set up and 180 pg template gDNA, corresponding theoretically to 1 target copy per reaction was added to the master mix. The master mix was pipetted into 60 wells. However, due to statistical variations, some wells had more than 1 target copy while others had none. The calibration curve was used to determine the actual number of copies within each of the 60 wells. [B] The expected number of copies per well was calculated theoretically using Poisson's equation and compared to the actual number of copies determined using the calibration curve. There was a high concordance between the single-copy number results obtained using the calibration curve and Poisson's equation, demonstrating the high sensitivity and robustness of the QuantiNova Probe PCR Kit.
Expression of EGFR in HeLa cells was analyzed. RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit. Reactions were run in triplicate using 10-fold dilutions of the cDNA (10 ng to 0.01 ng) on various cyclers fully exploiting the fast-cycling capabilities of each cycler. The QuantiNova Probe PCR Kit provides consistent sensitivity, reproducibility, and efficiency on the tested cyclers, despite the varying cycling conditions.
The performance of the QuantiNova Probe PCR Kit was compared to a probe PCR kit from supplier B on a Bio-Rad CFX Connect cycler [A] and a probe PCR kit from supplier L [B] on a ViiA7 cycler. Reactions were run in triplicate using 10-fold dilutions of plasmid DNA (106–102 copies per reaction) and a TaqMan assay detecting a 500 bp amplicon and using a minor-groove binding probe. The QuantiNova Probe PCR Kit provides significantly lower CT values, higher reproducibility, and higher reaction efficiency, compared to the probe PCR kits from suppliers B and L.
Duplex and singleplex PCR was performed on a Bio-Rad CFX96 cycler using TaqMan Assays for GAPD and TNF with the QuantiNova Probe PCR Kit. Ten-fold dilutions of leukocyte DNA (from 100 ng to 10 pg) were used as templates and reactions were run in triplicate. [A] Overlay of the TNF amplification curve for the singleplex and duplex reactions and [B] overlay of the GAPD amplification curve for the singleplex and duplex reactions. The plots demonstrate the comparability and reliability of the results for singleplex and duplex amplification using the QuantiNova Probe PCR Kit.
QuantiNova DNA Polymerase is kept in an inactive state by QuantiNova Antibody and QuantiNova Guard until the initial heat activation step.
The QuantiNova Probe PCR Master Mix contains an inert blue dye that turns green on addition of the template nucleic acid diluted in the QuantiNova Yellow Template Dilution Buffer, providing a visual indication of correct pipetting.