Introduction
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a method used to quantify nucleic acids in samples [1]. However, this approach often requires samples to undergo nucleic acid extraction which can be laborious, time consuming, and can result in the loss of precious samples. Experiments can be hindered further when reactions are inefficient due to a poorly designed master mix. Here, we highlight the newly formulated PrimeTime One-Step 4X Broad-Range Master Mix. This Master Mix has been specifically designed by IDT researchers for one-step RT-qPCR reactions. The 4X mixture of the mutant hot-start reverse transcriptase, mutant hot-start DNA polymerase, dNTPs and buffer provides high-quality performance for analysis of RNA. In developing this Master Mix, IDT researchers tested over 35,000 buffer components and selected the ideal mixture, generating a high-quality, flexible, and reliable master mix. The included enhancer solution was specifically designed to overcome inhibitors and improve amplification when using crude samples.PrimeTime One-Step 4X Broad-Range Master Mix
Amplify RNA from crude or purified nasopharyngeal and saliva samples
The PrimeTime One-Step 4X Broad-Range Master Mix can consistently and reliable amplify from either crude or purified nasopharyngeal (NP) and saliva samples (Figure 1). In this experiment, we amplified both NP and saliva samples that were incubated in viral transport medium (VTM) with ~10 copies of Accuplex SARS-CoV-2 reference material. These samples were then used crude or subject to an RNA extraction with the Applied Biosystems™ MagMax™ II Viral/Pathogen Isolation Kit (MVP II). The results shown in Figure 1 clearly illustrate the utility of this Master Mix to give you the flexibility to use either a direct amplification method or a purified sample method for amplification.
![PrimeTime One-Step 4X Broad-Range Master Mix results in comparable amplification of viral analytes between extracted and crude human saliva and nasopharyngeal samples.]( "22_QP_Figures_PrimeTime One-Step 4X Broad-Range MM_saliva and nasopharyngeal samples copy")
Figure 1. PrimeTime One-Step 4X Broad-Range Master Mix results in comparable amplification of viral analytes between extracted and crude human saliva and nasopharyngeal samples. RT-qPCR assays containing probes and primers specific for FluA, FluB, SARS-CoV-2, and RNase P were used to amplify 30 copies of AccuPlex™ SARS-CoV-2, Flu A/B and RSV Verification Panel (SARS-CoV-2 data shown) material that had been extracted (dark blue) or directly amplified (light blue). The AccuPlex™ analyte verification panel was first diluted into negative human saliva (A) or human nasopharyngeal samples (B) matrix prior to RNA extraction or direct specimen amplification, and RNA extractions were performed automated using the KingFisher™ Flex 96 and MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit. Reactions were amplified and identified using the QuantStudio™ 7 Flex instrument according to the manufacturer's instructions. Experiments were performed with multiple replicates (n = 20) for each condition.
Consistent and reliable multiplex amplification reaction
The PrimeTime One-Step 4X Broad-Range Master Mix is ideal for multiplex qPCR. Figure 2 shows amplification of a 5-plex reaction, demonstrating the master mix’s ability to handle these types of challenging experiments. Here we amplified 5 targets: CHEK1 (ROX), CSK (Tamara), HPRT (FAM), RNAse P (Cy5) and SFRS9 (SUN). We amplified all the targets over 8 logs of RNA template with a reaction efficiency between 94.9% and 105.1% (Figure 2).
![PrimeTime One-Step 4X Broad-Range Master Mix provides multiplexed (5-plex) RNA detection over 8 logs of RNA template]( "22_QP_Figures_PrimeTime One-Step 4X Broad-Range MM_exceptional multiplexed _5-plex_ RNA identification over 8 logs of RNA template copy")
Figure 2. PrimeTime One-Step 4X Broad-Range Master Mix provides multiplexed (5-plex) RNA detection over 8 logs of RNA template. RT-qPCR assays containing probes and primers specific for CHEK1 (A), CSK (B), HPRT (C), RNase P (D), and SFRS9 (E) genes were used to amplify a 10-fold dilution series of pooled RNA Ultramer templates ranging from 108 copies down to 10 copies. RT-qPCR reactions were run on the QuantStudio 7 Flex (n = 3).
Lower Ct values and higher end-point fluorescence than other vendors
The PrimeTime One-Step 4X Broad-Range Master Mix was challenged against other comparable commercially available master mixes. As seen in Figure 3, the PrimeTime One-Step 4X Broad-Range Master Mix shows both lower Ct and higher end-point fluorescence in comparison to other commercially available master mixes running the same assays. Assays for Influenza A (InfA, shown), Influenza B (InfB), SARS-CoV-2 and RNAse P were run with direct amplification from samples containing 30 copies of viral specimens in both human NP and human saliva samples. The PrimeTime One-Step 4X Broad-Range Master Mix reliably detected low copy numbers with direct amplification on unpurified samples and provided confidence in data analysis with high end-point fluorescence, particularly at later cycles.
![PrimeTime One-Step 4X Broad-Range Master Mix provides lower Ct and higher endpoint fluorescence from human saliva and nasopharyngeal matrix as compared to competitors]( "22_QP_Figures_PrimeTime One-Step 4X Broad-Range MM_saliva and nasopharyngeal matrix as compared to competitors")
Figure 3. PrimeTime One-Step 4X Broad-Range Master Mix provides lower Ct and higher endpoint fluorescence from human saliva and nasopharyngeal matrix as compared to competitors. RT-qPCR assays containing probes and primers specific for Influenza A (shown), Influenza B, SARS-CoV-2, and RNaseP genes were used to amplify 30 copies of pooled viral analytes spiked into negative human nasopharyngeal (A) or saliva (B) matrix stored in viral transport medium. RT-qPCR reactions were run on the QuantStudio 7 Flex (n = 6).
Lowering the limit of identification
Limit of identification data was generated using Ultramers™ RNA Oligonucleotides as a template. Ultramers were diluted from 100 copies/rxn down to 1 copy per reaction. As can be seen Figure 4, the PrimeTime One-Step 4X Broad-Range Master Mix shows excellent results down to 5 copies per reaction.
![PrimeTime One-Step 4X Broad-Range Master Mix provides low multiplexed limit of detection (LoD) when using purified RNA as template]( "22_QP_Figures_PrimeTime One-Step 4X Broad-Range MM_DECODED")
Figure 4. PrimeTime One-Step 4X Broad-Range Master Mix provides low multiplexed limit of detection (LoD) when using purified RNA as template. (A) RT-qPCR assays containing single-plex or multi-plexed probes and primers specific for InfA, InfB, SARS-CoV-2, and RNase P genes were used to amplify 100, 50, 10, 5, or 1 copies per reaction of purified RNA template. (B) A full analysis of results with a representative amplification plot (multiplexed SARS-CoV-2). RT-qPCR reactions were run on the QuantStudio 7 Flex (n = 21).
Conclusions
A reliable, flexible, and dependable master mix is critical for RT-qPCR approaches that generates powerful quantitative data. The PrimeTime One-Step 4X Broad-Range Master Mix can be used with samples that have not been extracted. Further the data presented above illustrates the utility of this master mix in multiplexed reactions as well as its ability to accelerate your RT-qPCR method by generating lower Ct values and higher endpoint fluorescence than other vendors.
