Tenfold serial dilutions of RNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for ILR2 (interleukin 1 receptor, type II). One-step RT-PCR was performed using the QuantiTect Probe RT-PCR Kit (PCR efficiency: 104%).
Total HeLa RNA (10 ng/rnx) samples were spiked with increasing amounts of human genomic DNA. Expression of MGAT1 was analyzed by qRT-PCR. MGAT1 is a single-exon gene that does not allow the discrimination of RNA and gDNA by assay design. Without the gDNA reduction step, Cq values decreased linearly, falsely indicating an increase in expression rate. In contrast, the expression levels remained similar when using the gDNA reduction step.
At ambient temperature the HotStaRTScript is inhibited by the RT-Blocker and the QuantiNova DNA Polymerase is kept inactive by QuantiNova Antibody and QuantiNova Guard. At 45°C the RT is activated while the QuantiNova DNA polymerase remains inactive. At 95°C the RT enzyme is denatured and the DNA polymerase is activated.
Real-time RT-PCRs including master mix, template RNA, primers and probes for [A] GAPDH, [B] MYC and [C] HSP90AA1 were left at room temperature (22°C) for 1 h and 2 h. These samples were run in parallel to freshly prepared reactions (set up on ice) on the Bio-Rad CFX 384 instrument. Duplicate reactions of 3 different template amounts were tested. The Cq values provided by the QuantiNova Probe RT-PCR Kit did not vary after extended storage of the samples at room temperature.
The performance of the QuantiNova Probe RT-PCR Kit was compared to a probe RT-PCR kit from supplier B on [A] a Bio-Rad CFX Connect cycler and [B] a probe RT-PCR kit from supplier L on a ViiA7 cycler. Reactions were run in duplicate using 10-fold dilutions of HeLa total RNA (100 ng – 10 pg) using TaqMan® assays for [A] MYC or [B] CDKNB1. Results show a high correlation between kits for higher template amounts; however, sensitivity was higher with the QuantiNova Probe RT-PCR Kit at 100 pg and 10 pg of total RNA (red arrows) compared to kits from supplier B and L.
The master mix contains an inert blue dye. Combined with QuantiNova Yellow Template Dilution Buffer, the resulting solution turns green, indicating that the reaction was set up correctly.