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Fast RT RT-PCR | Fast cDNA synthesis for qPCR

FAST cDNA SYNTHESIS FOR RT RT-PCR

fast RT-qPCR banner

About the Figure: Analyzing plant viral evolution using fast RT RT-PCR. To understand natural selection events during the evolution of viruses, researchers examined the fitness effects of mutations on a plant virus across several host species (including tobacco, shown in color in the figure above). The One Step SYBR® PrimeScript™ RT-PCR Kit II was used for fast RT-qPCR. Lalic, et al. (2011) PLoS Genetics 7(11): e1002378.

Accelerated cDNA Synthesis for Gene Expression Analysis

Stop waiting 30 to 60 minutes to synthesize cDNA templates for RT RT-PCR (real-time reverse-transcription PCR). With PrimeScript™ Reverse Transcriptase:
• Reverse transcription reactions can be completed in as few as 15 minutes.
• Or use a one-step RT RT-PCR PrimeScript kit and set your reverse transcription step to just 5 minutes.

PrimeScript Reverse Transcriptase is not only fast, it's 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.

  Try a sample of SapphireAmp Fast PCR Master Mix
 

Experimental Example: Rapid and Efficient Preparation of Template cDNA for RT RT-PCR with PrimeScript Reverse Transcriptase

Fast cDNA synthesis data

Figure 1. Real-time PCR efficiency using template cDNA synthesized with various RT reaction times. cDNA was synthesized from total RNA (2 pg-2 µg) using a RT time of either 15, 30, or 60 minutes. The resulting cDNA was used as a template for real-time PCR amplification of the mouse Actb gene. The resulting amplifcation curves and standard curve are shown.
  Cut reverse transcription reaction time from 60 minutes down to 15 minutes and generate the same reliable real-time PCR results. For maximal time savings, use cDNA synthesized with PrimeScript Reverse Transcriptase in combination with Takara fast real-time PCR master mixes, available for either SYBR® Green or TaqMan® detection.
In Figure 1, reverse transcription times for preparing cDNA template for real-time PCR analysis were compared. cDNA was synthesized from mouse total RNA (2 pg- 2 µg) using the PrimeScript RT Reagent Kit (Perfect Real Time) with an reverse transcription incubation step (37°C) of either 15, 30, or 60 minutes. After reverse transcription, 2 µl of cDNA was used for real-time PCR amplification of the mouse Actb gene using SYBR® Premix Ex Taq. Nearly identical results were obtained for all of the reverse transcription reaction times tested, indicating that 15 minutes was sufficient for preparing cDNA templates for real-time PCR analysis. For full experimental details, download the Application Note.

 

Experimental Example: Five Minute cDNA Synthesis for One-Step Real-Time PCR

One-step real-time RT-PCR

Figure 2. One-step real-time RT-PCR measurement of human. To detect HPRT1 mRNA, human liver total RNA (from 6.4 pg-100 ng) was used for one-step RT RT-PCR with HPRT1-specific primers. After the real-time PCR amplification, the Ct values from the amplification curves (top) were used to create the standard curve (bottom).
 

Reduce reverse transcription time and pipetting with Takara One-Step PrimeScript RT-PCR Kits. Available for both SYBR® Green and TaqMan® detection, these kits allow all RT 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, one-step RT RT-PCR was used to analyze HPRT1 expression (encodes hypoxanthine phosphoribosyl transferase1) in human liver total RNA (2 pg- 2 µg). The One-Step SYBR® PrimeScript RT-PCR Kit II (Perfect Real Time) was used for single-tube cDNA synthesis and real-time PCR with the following reaction conditions:

RT 42°C 5 min.
95°C 10 sec.
 
PCR 95°C 5 sec.
60°C 30 sec.
x40
Melt 60°C to 94°C  

The results (Figure 2) indicate that, with a 5 minute reverse transcription step, the One-Step SYBR® PrimeScript Kit allowed high sensitivity and specificity detection of HPRT1 mRNA across a wide range of template RNA concentrations.

 

 

RNA to Real-Time Data in Under 45 Minutes with One-Step Kits for TaqMan® Detection

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. This 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 1. Comparison of one-step real-time RT-PCR protocols for Takara One-Step PrimeScript RT-PCR Kit or similar kits from Companies A and Q

TakaraCompany ACompany Q
Stage 1
(RT)
42°C 5 min. 48°C 15 min. 50°C 10 min.
Stage 2 95°C 10 sec.
(Denature)
95°C 10 min.
(Activation)
95°C 5 min.
(Activation)
Stage 3 95°C 3 sec.
60°C 25 sec.
x40
95°C 15 sec.
60°C 60 sec.
x40
95°C 10 sec.
60°C 30 sec.
x40
Total Run Time (min.)4111460

 

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