Fast synthesis of cDNA templates for real-time RT-PCR
PrimeScript Reverse Transcriptase – a fast, robust solution
Ordinarily, it can take 30 to 60 minutes to synthesize cDNA templates for real-time RT-PCR (reverse-transcription PCR). With PrimeScript Reverse Transcriptase, RT reactions can be completed in as few as 15 minutes. Not fast enough? Use a one-step PrimeScript kit and set your reverse transcription step to just 5 minutes.
PrimeScript Reverse Transcriptase is not only fast, it is robust and can synthesize long, full-length cDNA molecules (up to 12 kb) with high specificity, accuracy, and yield. To learn more about PrimeScript Reverse Transcriptase, download the Application Note.
Reduce reverse transcription reaction time from >60 minutes to 15 minutes and generate the same reliable real-time PCR results. The PrimeScript RT Reagent Kit (Perfect Real Time) is optimized for fast, efficient synthesis of cDNA templates for real-time PCR.
When compared with a first-strand cDNA synthesis kit from Company L, the PrimeScript RT Reagent Kit synthesized cDNA templates from human total RNA with a higher RT efficiency and provided a significant time-savings benefit (Figure 1 and Table I). The PrimeScript RT Reagent kit provided higher yields of cDNA, resulting in lower Ct values (Table I). In addition, the PrimeScript kit provided a significant time savings, synthesizing real-time PCR-ready cDNA templates in just 15 minutes as opposed to over an hour as was required for the Company L kit.
Figure 1. Expression analysis of GAPDH and RNase P in human placenta total RNA. cDNA was synthesized using either PrimeScript or Company L RTase. The resulting cDNA was used for TaqMan®-based qPCR analysis. Amplification curves were obtained from all reactions, confirming successful reverse transcription and amplification of targets. However, the efficiency of reverse transcription was better for the PrimeScript reverse transcription reaction, as indicated by lower Ct values for both targets (Table I).
Table I. Ct values obtained from real-time PCR amplification.
|Target||Ct||Ct Mean||Ct Std Dev|
Available for both SYBR® Green and TaqMan detection, One-Step PrimeScript RT-PCR Kits allow all RT-PCR steps to be performed in a single tube, simplifying reaction set-up, minimizing the risk of contamination, and, with a reverse transcription step of just 5 minutes, allowing rapid analysis. In Figure 2, the One-Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) was used to analyze HPRT1 expression in human liver total RNA (2 pg–2 µg) in a single-tube reaction (cDNA synthesis and real-time PCR performed in tandem). This one-step protocol using PrimeScript RTase allowed high sensitivity and specificity detection of HPRT1 mRNA across a wide range of template RNA concentrations (Figure 2).
Figure 2. One-step real-time RT-PCR measurement of human HPRT1. Human liver total RNA (from 6.4 pg–100 ng) was used for one-step real-time RT-PCR with HPRT1-specific primers using the One-Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time). After the real-time PCR amplification, the Ct values from the amplification curves (top left panel) were used to create the standard curve (bottom right panel). The levels of HPRT1 target cDNA detected were highly consistent with the amount of total RNA input. The consistent melting curves show that the same amplification products were generated even when different amounts of template were used. The data generated a linear standard curve across the wide range of RNA template concentrations.
Compared to other commercially available one-step real-time PCR kits, the One-Step PrimeScript RT-PCR Kit (Perfect Real Time) protocol is significantly faster (Table II). The premix contains both PrimeScript Reverse Transcriptase, which is compatible with a 5 minute reverse transcription step, and TaKaRa Ex Taq HS polymerase, a high efficiency hot start PCR enzyme that reduces PCR amplification time.Table II. Comparison of real-time RT-PCR time* when using the One-Step PrimeScript RT-PCR Kit or similar kits from Companies A and Q.
|PrimeScript Kit||Company A||Company Q|
|Reverse transcription||5 min at 42°C||15 min at 48°C||10 min at 50°C|
|Denaturation/Activation (95°C)||10 sec
Annealing/Extension (60°C) Cycles
|Approx. total run time (min)||41||114||60|
* All reactions were run on an Applied Biosystems 7500 Fast Real-Time PCR system.
For the experimental example in Figure 1, one microgram of human placenta total RNA was reverse transcribed using random hexamers in a total reaction volume of 20 µl according to the recommended reaction composition and reaction conditions. The conditions for the PrimeScript RT reaction are as follows:
|5X PrimeScript Buffer (for Real Time)||4 µl|
|PrimeScript RT Enzyme Mix I||1 µl|
|Random 6 mers (100 µM)||2 µl|
|Total RNA (1µg/µl)||1 µl|
|RNase Free dH2O||12 µl|
Reactions were performed according to the recommended conditions:
|PrimeScript Kit||Company L Kit|
|37°C, 15 min||65°C, 5 min|
|85°C, 5 sec||25°C, 10 min|
|4°C, hold||50°C, 50 min|
|85°C, 5 min|
|Total: approx. 15min||Total: approx. 70 min|
After reverse transcription, the RT products were diluted with an equal volume of dH2O, and 2 µl was used for real-time PCR (corresponds to 50 ng total RNA/PCR reaction). Custom TaqMan assays were used to analyze expression of GAPDH and RNase P using an ABI 7900HT Fast Real Time PCR System. All reactions were performed in duplicate. The PCR conditions were 95°C for 10 min, then 40 cycles of 95°C for 15 sec and 55°C for 1 min. Data was analyzed using SDS v2.4 software automated analysis.
For the experimental example in Figure 2, the One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) was used to measure human HPRT1 by SYBR Green-based one step real-time quantitative PCR. Human liver total RNA (6.4 pg–100 ng) and sterilized water (negative control) were used as templates with HPRT1 primers. Reactions were prepared according to the recommended protocol and were run on a Thermal Cycler Dice Real Time System.
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