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AccuPower RocketScript Cycle RT PreMix is a ready-to-use lyophilized mastermix containing all components for first-strand cDNA synthesis from purified Poly(A) or total RNA template.
The premix contains Gentaur's exclusive M-MLV based thermostable reverse transcriptase (RTase), RocketScript, and oligo dT20 for added convenience. Conditions are optimized for Gentaur's patented Cyclic Temperature Reverse Transcription (CTRT) in a premix form.
Native M-MLV RTase has maximum activity at relatively low temperatures (42°C), causing several problems in reverse
transcription of RNA molecules with complex secondary structure. AccuPower RocketScript Cycle RT PreMix has thermostable activity across a wide temperature range, (42°C – 70°C), allowing efficient cDNA synthesis from virtually any RNA.
The lyophilized PreMix contains all components necessary for a successful CTRT reaction, including RTase, RNase inhibitor and buffer components. Just add template RNA, primers and water, and the RT reaction is ready to go.
Schematic representation of the 5' UTR of a gene, with complex secondary structure, at three different temperatures. Note that RocketScript shows full activity at 70°C allowing it to synthesize the complete gene sequence where M-MLV and other Reverse Transcriptase's fail.
Features and Benefits
Thermostable Activity: RocketScript is able to perform reverse transcription reactions throughout a wide range
of temperatures from 42°C to 70°C.
Enhanced Performance: RocketScript has enhanced performance to handle high and low input RNA concentrations
as well as short and long RT target sizes.
Easy-to-use: The product contains the enzyme itself, plus RNase inhibitors and all other components necessary for the best reverse transcription results in the tube.
Reproducibility: Gentaur's strict quality controlled production system ensures that your results will be reproducible experiment after experiment.
Flexible Reaction Conditions: Both CTRT and first-strand cDNA synthesis are both possible, with reaction temperatures
ranging from 42°C to 70°C.
Convenient: Just add template and primers and start your reaction. dNTPs, buffer and enzyme are provided.
Stability: Stable at room temperature for a month one year at 4°C and for 2 years in a -20°C freezer
RNase, DNase and Proteinase-free: Ensures the integrity of your samples.
Broad range of working temperatures: For RNAs that are G:C rich or significant secondary structure
Stable for 2 years at -20°C: Long shelf life
Specifications
5' to 3' exonuclease: No
3' to 5' exonuclease: No
3’ – A Overhang: No
Fragment Size: Up to 10 kb
Application
- First-strand synthesis of cDNA from RNA molecules (Reverse Transcription)
- RT-PCR
- Random priming reaction
- Library construction
- Probe labeling
- mRNA 5’end mapping by primer extension analysis
- Real time PCR
Experimental data
Figure 1. Comparison of amplification efficiency between AccuPower RocketScript Cycle RT PreMix and competitors’ RTases
(a) Sensitivity test
Primer set: Human TFRC set
Lane M: 1 kb DNA Ladder
Lane1: 100 ng Human total RNA from HeLa cell
Lane 2: 10 ng Human total RNA from HeLa cell
Lane3: 1 ng Human total RNA from HeLa cell
Lane4: 100 pg Human total RNA from HeLa cell
RT reaction condition is performed according to each manufacturer’s recommendations
(b) Full-Length cDNA synthesis test
RT reactions were performed according to each manufacturer’s recommendation. All cDNAs were amplified with AccuPower Hotstart PCR Premix (K-5050) from Gentaur
Note: Competitor products show inhibition at high input concentrations of total RNA
Lane 1: 1 μg Human total RNA from HeLa cell
Lane 2: 100 ng Human total RNA from HeLa cell
Lane 3: 10 ng Human total RNA from HeLa cell
Figure 2. Complex RNA amplification results of RocketScript Cycle RT PreMix
Each target gene was amplified after performing reverse transcription with AccuPower RocketScript Cycle RT PreMix.
Reverse transcription conditions: Conventional 1 hr incubation at 42°C, 50°C, or 60°C, deactivation at 95°C for 5 min
A: M-MLV Reverse Transcriptase
B: AccuPower RocketScript Cycle RT PreMix with Oligo (dT)20
Lane 1: 100 ng Human total RNA from HeLa cell
Lane 2: 10 ng Human total RNA from HeLa cell
| Concentration (copies/rxn) | FTRT (Ct) | CTRT with 1 cycle (Ct) | CTRT with 10 cycles (Ct) |
| 10,000 | 19.46 | 18.77 | 18.51 |
| 1,000 | 24.11 | 24.04 | 22.93 |
| 100 | 29.78 | 28.35 | 28.19 |
| 10 | 32.87 | 33.00 | 31.05 |
Figure 3. Low copy species enrichment by cycle.
Comparing FTRT (Fixed Temperature Reverse Transcription) to 1 and 10 cycle(s) of CTRT reveal progressive improvement in detected cDNA yield as input copies decrease.
FTRT: 60 min incubation at 50°C followed by 5 min deactivation at 95°C
CTRT: Cycles of 37°C annealing 10 sec, 50°C cDNA synthesis 4 min, 55°C secondary structure melting and cDNA synthesis
30 sec
Primer set: Human GAPDH
Human Total RNA from HeLa cells
qPCR with AccuPower GreenStar qPCR PreMix (K-6210)
Figure 4. Amplification comparison by cycle
FTRT (Fixed Temperature Reverse Transcription) conditions:
| Step | Temperature | Time | No. of Cycles |
| dT20 | |||
| cDNA synthesis | 50°C | 60 min | 1 |
| Heat inactivation | 95°C | 5 min | 1 |
CTRT (Cyclic Temperature Reverse Transcription) conditions:
| Step | Time | Temperature | No. of Cycles |
| dT20 | |||
| Primer annealing | 37°C | 10~30 sec | 1, 5, 10, or 15 |
| cDNA synthesis | 50°C | 4 min | |
| Melting secondary structure & cDNA synthesis | 55°C | 30 sec | |
| Heat inactivation | 95°C | 5 min | 1 |
Primer set: Human GAPDH and myc set
Lane M: 1 kb DNA Ladder
Lane1: 10 ng Human Total RNA from HeLa Cell
Lane 2: 1 ng Human Total RNA from HeLa Cell
Lane 3: 100 pg Human Total RNA from HeLa Cell
Lane 4: 10 pg Human Total RNA from HeLa Cell
Figure 5. Comparison of amplification results between Gentaur RocketScriptTM Cycle RT PreMix and competitor RTases
All cDNAs were amplified with AccuPower Hotstart PCR Premix (K-5050) from Gentaur
Primer set: human Myc498 bp set
Lane M: 1 kb DNA Ladder
Lane1: 10 ng Human total RNA from HeLa Cell
Lane 2: 1 ng Human total RNA from HeLa Cell
Lane 3: 100 pg Human total RNA from HeLa Cell
Lane 4: 10 pg Human total RNA from HeLa Cell
