QuantiFast Multiplex Kits reduce total PCR run time by up to 50% in real-time two-step RT-PCR (40 cycles run; comparison with QuantiTect Multiplex Kits). I: iCycler iQ; L1: LightCycler 480; L2: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500 Fast System; M: Mx3005P.
Duplicate reactions were run on the Applied Biosystems 7500 Fast System using a DNA template mix providing 108 copies of β-actin (data shown in insets) and 106 to 10 copies of RPS27A (a ribosomal protein). The [A] QuantiFast Multiplex PCR +R Kit showed clearly higher sensitivity than the [B] duplex PCR kit from Supplier AII, enabling the cycler in fast-cycling mode to detect 10 copies of target and quantify over 6 log dilutions of template.
Triplex and singleplex PCR were carried out on the LightCycler 480 using the QuantiFast Multiplex PCR +R Kit and Primer Express designed TaqMan assays for [A] RPS27A (a ribosomal protein), [B] GAPDH (a housekeeping gene), and [C] UBC (a housekeeping gene). The template was HeLa cell cDNA (10 ng, 1 ng, or 0.1 ng), and reactions were run in duplicate. The comparable CT values for triplex PCR (colored curves) and singleplex PCR (gray curves) demonstrate the reliability of triplex PCR with the QuantiFast Multiplex PCR +R Kit when analyzing targets of differing abundance.
4-plex, real-time PCR was carried out using the QuantiFast Multiplex PCR +R Kit and Primer Express designed TaqMan assays on the Applied Biosystems 7500 Fast System. Triplicate reactions were run using 10 ng Ramos cell line cDNA.
[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.
Duplex and singleplex PCR were carried out using the QuantiFast Multiplex PCR +R Kit and assays for the t(8;14) chromosomal translocation and for GAPDH on the Applied Biosystems 7500 Fast System. Quadruplicate reactions were run using genomic DNA from the Ramos cell line as template (twofold dilutions from 10 ng to 0.625 ng). CT values increased linearly by 1 CT value with decrease in template dilution for both the [A] singleplex and [B] duplex reactions, demonstrating the ability of the kit to precisely discriminate between small differences in template amount.