DTI FabScript Reverse Transcriptase

Reduce reverse transcription reaction time from >60 minutes to 15 minutes and generate the same reliable real-time PCR results. The FabScript 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 FabScript RT Reagent Kit synthesized cDNA templates from human total RNA with a higher RT efficiency and provided a significant time-savings benefit (Figure 1; Table I). The FabScript RT Reagent Kit provided higher yields of cDNA, resulting in lower Ct values (Table I), and synthesized real-time PCR-ready cDNA templates in just 15 minutes as opposed to the Company L kit, which required more than one hour.

Available for both green intercalating dye- and probe-based detection, DTI FabScript RT-PCR kits allow all RT-PCR steps to be performed in a single tube, simplifying reaction setup, minimizing the risk of contamination, and, with a reverse transcription step of just 5 minutes, allowing rapid analysis. In Figure 2, the One-Step TB Green 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 FabScript RTase allowed high sensitivity and specificity detection of HPRT1 mRNA across a wide range of template RNA concentrations (Figure 2).

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-min reverse transcription step, and Takara Ex Taq HS polymerase, a high efficiency hot-start PCR enzyme that reduces PCR amplification time.

* 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 FabScript RT reaction were as follows:

Reactions were performed according to the recommended conditions:

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, followed by 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 TB Green PrimeScript RT-PCR Kit II (Perfect Real Time) was used to measure human HPRT1 by intercalator-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 Dice Real Time System thermal cycler.

FabScript Reverse Transcriptase is fast, robust, and can synthesize long, full-length cDNA molecules (up to 12 kb) with high specificity, accuracy, and yield. It helps reduce the time it takes to synthesize cDNA templates for real-time RT-PCR from 30–60 min to under 15 min.