PrimeTime One-Step RT-qPCR Master Mix is a one-step qPCR master mix that offers excellent capabilities for standard gene expression qPCR experiments.
The PrimeTime One-Step RT-qPCR Master Mix is a ready-to-use, 2X concentrated master mix that is designed for use in probe-based, real-time quantitative PCR. The PrimeTime One-Step RT-qPCR Master Mix contains an antibody-mediated hot-start DNA polymerase, reverse transcriptase, dNTPs, MgCl2, enhancers, and stabilizers.
Figure 1. PrimeTime One-Step RT-qPCR Master Mix identifies SFRS9 RNA in as little as 2 pg of total human RNA. A PrimeTime qPCR assay containing probes and primers designed for SFRS9 was used to amplify a dilution series of SFRS9 RNA Ultramer™ template to create a standard curve (1010 to 10 copies) (black line), and then a 10-fold dilution series of total RNA from HEK293 cells (100 ng to 1 pg) (rainbow dots) (n = 3).
Figure 2. PrimeTime One-Step RT-qPCR Master Mix provides high functionality in multiplex assays. Multiplex qPCR assays containing probes and primers designed for HPRT-FAM, CSK-ATTO™ 647 (ATTO-TEC GmbH), SFRS9-SUN™, and CHEK1-ROX amplified a dilution series of pooled RNA Ultramer templates to create standard curves for each gene. The amount of each transcript in 1 ng of HEK293 total RNA (●) was determined. For each gene, an R2 = 1 and efficiency between 100.9– 103.1% was obtained. Assays were performed on a QuantStudio™ 7 Flex qPCR instrument (n = 3).
Figure 3. PrimeTime One-Step RT-qPCR Master Mix has no loss of efficiency and identification capabilities in fast qPCR cycling parameters. Multiplexed qPCR sets containing probes and primers designed for HPRT-FAM and SFRS9-SUN were used to amplify a dilution series of pooled HPRT and SFRS9 RNA Ultramer templates to create a standard curve (1010 to 10 copies) using either standard [15 min. 50°C; 3 min. 95°C; 40 x (15 sec. 95°C, 1 min. 60°C] or fast [15 min. 50°C; 3 min. 95°C; 40 x (5 sec. 95°C, 30 sec. 60°C] qPCR cycling parameters. For each standard curve the R2 = 1 and the efficiency was between 97.1–101.1% demonstrating that there was no loss of function in fast cycling conditions (n = 3).
Figure 4. PrimeTime One-Step Master Mix provides exceptional low copy number RNA identification in comparison to alternate master mixes. RT-qPCR sets containing probes and primers designed for HPRT-FAM amplified a 10-fold dilution series of FAM RNA Ultramer templates ranging from 1010 copies down to 10 copies. The lowest template concentrations were highlighted in these logarithmic amplification plots with the indicated color to emphasize the low RNA copy number identification capabilities of each master mix product. RT-qPCR reactions were run on the QuantStudio 7 Flex and cycling parameters were adjusted for each master mix product as per the manufacturer’s recommendations (n = 3).
The reference dye included with our master mixes is ROX.
We tested up to 15 freeze-thaw cycles, which is more extreme than typical laboratory usage. No detrimental effects or loss in Cq were observed. However, we still recommend following good laboratory practice by minimizing freeze-thaw cycles.
Depending on the stability of the RNA template, plates containing reaction mix and template can typically be stored at 4°C for up to 8 hours prior to the qPCR run without negatively affecting performance of the master mix.
The master mix can be used with either standard or fast cycling conditions, depending on what qPCR system you are using. For best results, use one of the cycling parameters provided in the PrimeTime One-Step RT-qPCR Master Mix protocol:
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.