QuantiFast Probe Kits reduce total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.
The QuantiFast Probe PCR +ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93% in two-step PCR on the Mastercycler ep realplex. Duplicate reactions (25 μl volume) were performed using 10-fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin (a regulatory protein). NTC: no template control.
Reactions were run in duplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan gene expression assay for IL1RN (a cytokine) on the ABI PRISM 7900. The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier AII (which was used according to the standard cycling protocol), providing much lower CT values and transcript quantification from down to 10 pg cDNA.
Reactions were run in triplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a Primer Express designed TaqMan assay for IL8 (a chemokine) on the ABI PRISM 7900. [A] The QuantiFast Kit showed greater sensitivity than [B] the kit from Supplier I (which was used according to the standard cycling protocol), providing lower CT values and transcript quantification from down to 0.01 ng cDNA.
Duplicate two-step RT-PCR reactions were run using a set of primers and FRET probes for b-2 microglobulin from Supplier R on the LightCycler 2.0. The amplification plots for the QuantiFast Kit show the kit's wide dynamic range.
A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.
