Researchers recognize real-time RT-PCR as the primary technique for quantifying RNA when analyzing differential gene expression, infectious disease etiology and environmental quality. GoTaq® RT-qPCR Systems use dye-based RNA quantification that does not require labeled primers or probes to detect amplification products and offer high specificity, broad linear range, sensitivity and reproducibility. These systems combine the benefits of GoScript™ Reverse Transcriptase and GoTaq® Hot Start Polymerase technology with optimized buffers for robust cDNA synthesis and qPCR. Amplified products are detected using Promega BRYT Green® Dye, a proprietary dye included in the reaction buffers.
We demonstrated the performance of the Promega GoTaq® 1-Step RT-qPCR System (Cat.# A6020) in standard curve assays using varied amounts of human total RNA (HT RNA) and 11 previously published PCR primer pairs (see Supplemental Information, Table 1). We amplified one of these targets, glyceradehyde-3-phosphate dehydrogenase (GAPDH) mRNA (GenBank® accession number NM_002046), to demonstrate consistent performance of the GoTaq® 1-Step RT-qPCR System using varied amounts of human total RNA (HT RNA). We also amplified GAPDH mRNA target to compare the performance of the GoTaq® 1-Step RT-qPCR System to that of the GoTaq® 2-Step RT-qPCR System (Cat.# A6010) and to 4 one-step RT-qPCR kits from other manufacturers. Experiments and comparisons were performed and reported in adherence to MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments, see Resources section).
Materials and Methods
We assessed the performance of the GoTaq® 1-Step RT-qPCR System by analyzing the expression levels of 11 differentially expressed mRNA targets, including several reference genes (see Supplemental Information, Table 1). We used one of these targets, GAPDH, to compare performance of the GoTaq® 1-Step RT-qPCR System to that of the GoTaq® 2-Step RT-qPCR System and four one-step RT-qPCR reagent systems from other manufacturers. These four reagent systems are referred to as Reagents A, I and B, which use double-stranded DNA-binding dyes, and Reagent T, which is a hydrolysis probe-based system. Reactions were performed using the protocols in the GoTaq® 1-Step RT-qPCR and GoTaq® 2-Step RT-qPCR System Technical Manuals (TM355 and TM337, respectively) or appropriate instructions provided by the other manufacturers.
For two-step RT-qPCR, we used HT RNA (QPCR Human Reference Total RNA, Stratagene Cat.# 750500) as a template for cDNA synthesis in a 20µl reaction as described in the GoTaq® 2-Step RT-qPCR System Technical Manual TM337. The amounts of HT RNA are indicated in the legend for Figure 5. Following reverse transcription and heat inactivation of the GoScript™ Reverse Transcriptase, cDNA was diluted in water prior to qPCR or added directly to qPCR without dilution, as indicated. We used a mixture of Random Primers (Cat.# C1181) and Oligo(dT)15 Primer (Cat.# C1101) at 25ng/μl each or GAPDH G5 reverse primer (see Supplemental Information, Table 1) as the reverse transcriptase primer, as indicated. No-template controls were performed for each set of amplifications.
For one-step RT-qPCR, we amplified HT RNA as described in the GoTaq® 1-Step RT-qPCR System Technical Manual TM355 in replicate 50µl, 25µl or 10µl reactions. Reactions were amplified and analyzed using the Applied Biosystems 7500 or 7500 FAST Real-Time PCR System in the Standard or Fast mode, as indicated. The amounts of HT RNA template were 100ng–100fg unless otherwise indicated. Reverse transcription conditions were 42°C for 15 minutes unless otherwise indicated. Following reverse transcription, the GoScript™ Reverse Transcriptase was inactivated and the hot-start DNA polymerase was activated at 95°C for 10 minutes, followed by 40 cycles of two-step qPCR (95°C for 15 seconds, 60°C for 1 minute) for Figures 4 and 5, Panel A, and Supplemental Information, Figure 1, or three-step qPCR (95°C for 15 seconds, 62°C for 35 seconds. 72°C for 25 seconds) for Figures 1–3, and dissociation analysis. No-template control reactions were performed for each set of amplifications. Two-step qPCR cycling was also used for the two-step RT-qPCR assays in Figure 5 (Panels B, C and D).
All RT-qPCR assays included 200nM of both forward and reverse primers except for the 100nM hydrolysis probe reactions in Supplemental Information, Figure 1. For two-step RT-qPCR assays, the reverse PCR primer concentration included the amount of reverse primer used for reverse transcription. Reactions performed without reverse transcriptase were used to confirm that the HT RNA was not contaminated with genomic DNA (data not shown). Except where noted, PCR was performed in triplicate using a reaction volume of 50µl.
Efficient Amplification and Consistent Performance
Performance of the GoTaq® 1-Step RT-qPCR System is exemplified in the ten- and twofold dilution series data shown in Figure 1. Standard curve analysis of triplicate reactions containing 300ng–10fg of HT RNA amplified with the G7/G8 GAPDH primer set demonstrated 97.6% amplification efficiency across a greater than 7-log range of HT RNA with excellent r2 values (r2 = 0.999; Figure 1, Panel A). Absence of nonspecific product was shown by the dissociation curves, which contained a single peak, and by the absence of amplification in no-template controls. Figure 1, Panel B shows amplifications of twofold dilutions of HT RNA across a 6-log range (100ng–381fg). The clear separation of groups of replicate reactions demonstrates that samples with concentration differences as low as twofold can be distinguished using the GoTaq® 1-Step RT-qPCR System. Figure 2, Panel A shows the performance of GAPDH amplifications in 50µl and 10µl reactions, and the tight clustering of 40 replicate reactions containing 10pg of HT RNA. Assay reproducibility was demonstrated by performing two runs with conditions identical to those in Figure 1, Panel A, but performed on two different days (compare Figure 2, Panels B and C) and a third run using the Applied Biosystems 7500 Real-Time PCR System in Fast mode in 25µl reactions (Figure 2, Panel D). Slopes, intercepts, and r2 values obtained in the three runs are virtually indistinguishable from those shown in Figure 1, Panel A, demonstrating the reproducible performance of the GoTaq® 1-Step RT-qPCR System.
Performance with a Broad Range of mRNA Expression Levels
Similar high-quality performance was observed with ten additional mRNAs transcripts, representing a broad spectrum of expression levels. Different mRNA targets were amplified in triplicate samples of serially diluted HT RNA (100ng–100fg). Each reaction was analyzed to determine specificity, linear range and efficiency. Figure 3 shows that the slope, r2 values and change in fluorescence (ΔRn) were equivalent for the most (GAPDH) and least abundant (UBC) transcripts in the RNA sample despite a large difference (~12 cycles) in Y intercept values. A summary of the amplification results for GAPDH (G7/G8) and 10 other targets of varying abundance is shown in Supplemental Information, Table 2. For each target, PCR efficiency was greater than 90%, and performance was consistent across the input RNA range as shown by the r2 values, all of which were equal to or greater than 0.99.
BRYT Green® Dye
The BRYT Green® Dye in the GoTaq® 1-Step RT-qPCR System is less inhibitory to polymerase than other double-stranded DNA (dsDNA) binding dyes such as SYBR® Green or SYBR® GreenER™ dyes. This property allows BRYT Green® Dye to be used at a higher concentration, resulting in overall brighter fluorescence. In some cases, the bright fluorescence enables real-time PCR instruments to detect amplification products earlier and report earlier Cq values relative to a common threshold. This is demonstrated in Figure 4, in the one-step quantification of GAPDH mRNA (G3/G5 target) in reactions containing serially diluted HT RNA (100ng–1pg) using the GoTaq® 1-Step RT-qPCR System, which contains CXR Reference Dye, on one half of the plate, and Reagents A, B or I on the other half. For reactions with Reagents A, B and I, the reference dye ROX™ was either present or added to a final concentration of 50nM for analysis and comparison of normalized fluorescence under the same conditions. For each run, we observed significantly brighter ΔRn (normalized change of fluorescence) and earlier Cq values in the GoTaq® 1-Step RT-qPCR reactions. The comparisons are illustrated by the sets of amplification curves shown in Figure 4.
Consistent Results Using One-Step or Two-Step RT-qPCR Protocols
One-step RT-qPCR is sometimes assumed to be less sensitive and specific than two-step RT-qPCR. When we compared one-step and two-step protocols, our data did not support this bias. For this comparison, GAPDH (G3/G5 target) was quantified in reactions containing cDNA template (corresponding to 100ng–100fg of HT RNA) synthesized using each of three optional GoTaq® 2-Step RT-qPCR cDNA synthesis methods, or in GoTaq® 1-Step RT-qPCR reactions containing samples of serially diluted HT RNA (100ng–100fg; Figure 5). The results demonstrate equivalent specificity and performance with sensitivity of as little as 100fg (the lowest amount of template that resulted in an amplification curve and Cq value), ≥90% efficiency, and ≥0.99 r2 values across the same range of template.
GoTaq® 1-Step RT-qPCR performed well in comparison to four other RT-qPCR reagent systems to detect GAPDH mRNA (G3/G5 target) in serially diluted (100ng–1pg) HT RNA (Supplemental Information, Figure 1). PCR slopes, intercepts and r2 values obtained with the GoTaq® 1-Step RT-qPCR System compared favorably with those obtained using dye-based systems (Reagents A, I and B) or a probe-based system (Reagent T). Dissociation curves for no-template controls amplified with the GoTaq® 1-Step RT-qPCR System and Reagents A and B did not contain peaks, indicating that no amplification products were synthesized. In contrast, the dissociation curves for two of three no-template controls amplified with Reagent I indicated the presence of minor, nonspecific products.
We have demonstrated that the GoTaq® 1-Step and 2-Step RT-qPCR Systems offer equivalent performance and sensitivity. Compared to competitor one-step RT-qPCR systems, the GoTaq® RT-qPCR Systems can offer greater total change of fluorescence and earlier Cq detection with minimal nonspecific amplification. As a result, GoTaq® 1-Step and 2-Step RT-qPCR Systems meet the diverse needs of researchers performing RNA quantification with dye-based analysis.