Tenfold dilutions of human leukocyte cDNA (10 ng to 10 pg) were used as template in 4-plex, real-time PCR. Reactions were run in triplicate using either the Rotor-Gene Q and Rotor-Gene Multiplex PCR Kit or an instrument and kit from Supplier S. [A] Targets amplified, and reporter dyes of corresponding TaqMan probes. [B] CT values obtained for all 4 targets (instrument and kit from Supplier S did not successfully detect IFNG; N.D.). Lower CT values on the Rotor-Gene Q demonstrate detection with greater sensitivity. [C] Amplification plots for TNF using the Rotor-Gene Multiplex PCR Kit. [D] Amplification plots for TNF using a kit from Supplier S. [E] Amplification plots for IFNG using the Rotor-Gene Multiplex PCR Kit. [F] Amplification plots for IFNG using a kit from Supplier S.
Duplex, real-time two-step RT-PCR was carried out on the Rotor-Gene Q using the Rotor-Gene Multiplex PCR Kit and self-designed TaqMan assays for [A] IL8 (interleukin 8) and [B] ACTB (β-actin). Analysis of tenfold dilutions of leukocyte cDNA template from 100 ng to 1 pg provided high PCR efficiencies of around 95%. [C] The CT values were comparable with those achieved in control singleplex reactions, demonstrating the reliability of the duplex assay.