QuantiFast Probe Kits reduce total RT-PCR run time by up to 60% in real-time one-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.
Reactions were run in duplicate using 10-fold dilutions of human leukocyte RNA (100 ng to 0.1 ng) and a Primer Express designed TaqMan assay for IL12RBI (a type I transmembrane protein) on the Applied Biosystems 7500 Fast System. [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 0.1 ng RNA.
Ubiquitin expression in human leukocytes was analyzed using a Primer Express designed TaqMan assay. Two-step and one-step qPCR reactions were run in duplicate on the ABI PRISM 7900. [A] 10-fold cDNA dilutions (100 ng to 10 pg) were analyzed using the QuantiFast Probe PCR Kit. [B] 10-fold RNA dilutions (100 ng to 10 pg) were analyzed using the QuantiFast Probe RT-PCR Kit.
Triplicate reactions were performed using 10-fold dilutions of human leukocyte RNA and a TaqMan assay for β-actin. The QuantiFast Probe RT-PCR Kit provided accurate gene expression analysis from 100 ng down to 1 pg of RNA template.