DTI FabScript Reverse Transcriptase
DTI FabScript Reverse Transcriptase
DTI FabScript Reverse Transcriptase is a modified MMLV (Moloney Murine Leukemia Virus) RTase. This enzyme has extremely high extension capability and can synthesize long first-strand cDNA efficiently. Even for difficult templates, including RNAs with complex secondary structure, it is possible to synthesize first-strand cDNA at the normal reverse transcription temperature (42℃) with this enzyme. It is not necessary to perform the RT reaction at higher temperatures (a condition that may cause RNA degradation). This enzyme is suitable for preparation of long cDNAs, for construction of cDNA libraries that include full-length cDNA, etc.
SKU:DT0301.10K
DTI FabScript Reverse Transcriptase
| Brand | Catalogue Number | Pack Size |
|---|---|---|
| DTI FabScript Reverse Transcriptase | DT0301.10K | 10000 U |
- Strong strand-displacement and extension capability capable of synthesizing long and full-length cDNA molecules (up to 12 kb).
- Preparation of cDNA at 42°C results in lower background and higher cDNA yields because the lower temperature does not promote RNA degradation.
- Outstanding accuracy lowest error rate among five commercially available reverse transcriptases tested.
- Highly specific low rates of non-specific annealing, even on incompletely denatured RNA.
- Works on challenging templates excellent results even with GC-rich templates and templates with high levels of secondary structure.
- Highly sensitive use less of your precious RNA samples.
- Demonstrated success with reverse transcription reaction times of 30 min (60 min for longer transcripts).
DTI FabScript Reverse Transcriptase - Protocol and Downloads
| Document | Download Link |
|---|---|
| DTI FabScript Reverse Transcriptase - Protocol | Download User Manual |
| DTI FabScript Reverse Transcriptase - Certificate of Analysis | Download CoA - DT0301.10K |
| DTI FabScript Reverse Transcriptase - Promotional Document | Download Flyer |
Results: Rapid and Efficient Preparation of Template cDNA for Real-Time PCR
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.
Figure 1: Expression analysis of GAPDH and RNase P in human placenta total RNA. cDNA was synthesized using either FabScript 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 FabScript 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 |
|---|---|---|---|
| FabScript GAPDH | 25.24 | 25.21 | 0.02 |
| Company L GAPDH | 26.26 | 26.28 | 0.01 |
| FabScript RNP | 30.21 | 30.18 | 0.03 |
| Company L RNP | 31.24 | 31.08 | 0.11 |
Reduce reverse transcription time and pipetting. 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).
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 TB Green PrimeScript RT-PCR Kit II (Perfect Real Time). After 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 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.
Table II: Comparison of real-time RT-PCR reaction times* when using the One-Step PrimeScript RT-PCR Kit or similar kits from Companies A and Q.
| FabScript 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 | 10 min (Activation) | 5 min (Activation) |
| PCR | Denaturation (95°C), Annealing/Extension (60°C) Cycles | 3 sec, 25 sec x40 | 15 sec, 60 sec x40 |
| Approx. total run time (min) | 41 | 114 | 60 |
* All reactions were run on an Applied Biosystems 7500 Fast Real-Time PCR system.
Methods
Figure 1 Methods: 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