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

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:

CTRT (Cyclic Temperature Reverse Transcription) conditions:

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 RocketScript^TM 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

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 (RT)
- RT-PCR
- Random priming reaction
- Library construction
- Probe labeling
- mRNA 5end Mapping by Primer Extension Analysis
- Real time PCR

Figure 1. Sensitivity comparison between AccuPower RocketScript RT PreMix and M-MLV RTase
Sensitivity results of AccuPower RocketScript RT PreMix using GAPDH compared with conventional Reverse transcriptases. 
Each 100 ng – 10 fg of total RNA used for RT and the same amount of RT products used for electrophoresis.
Lane 1: 10 fg Human total RNA from HeLa cell           Lane 2: 100 fg Human total RNA from HeLa cell
Lane 3: 1 pg Human total RNA from HeLa cell            Lane 4: 10 pg Human total RNA from HeLa cell
Lane 5: 100 pg Human total RNA from HeLa cell        Lane 6: 1 ng Human total RNA from HeLa cell
Lane 7: 10 ng Human total RNA from HeLa cell          Lane 8: 100 ng Human total RNA from HeLa cell

Figure 2. Comparison of amplification efficiency between AccuPower RocketScript RT PreMix and competitorsM-MLV RTase.
RocketScript is able to handle a wide range of sample concentrations and transcript lengths so your downstream applications are minimally effected by the reverse transcription step.
Lanes 1-3: 1,000 ng, 100 ng and 10 ng of total RNA from HeLa cells, respectively.

Figure 3. Sensitivity comparison between AccuPower RocketScript RT PreMix and competitor RTases using Real Time PCR
Reverse transcription conditions: conventional 1 hr incubation at 60°C, deactivation at 95°C for 5 min All cDNAs were amplified with AccuPower DualStar™ qPCR PreMix (K-6110) from Gentaur.

Concentration	RocketScript RT PreMix	Supplier Q	Supplier I
10,000	23.91	25.63	24.43
1,000	27.33	28.92	28.03
100	30.62	32.42	30.88
10	33.63	35.43	33.95
Efficiency	104%	103%	108%
Linearity	0.99999	0.9996	0.9995

Specifications

3' to 5' exonuclease: No
3’ – A Overhang: No
Fragment size: 9 kb

Application

cDNA synthesis

Figure 1. Reaction condition comparison of Cyclic Reverse Transcription versus conventional reverse transcription PCR. 800 ng of HeLa cell total RNA was reverse transcribed using different reaction conditions and then 800 bp, 1.5 kb, 2.0 kb, and 2.6 kb of human transferrin receptor gene were amplified using AccuPower PCR PreMix.

M: 1 kb DNA Ladder (D-1040)
Lane 1 - 4: Conventional RT with dT₂₀ at 42°C
Lane 5 – 8: Conventional RT with dN₁₂ at 42°C
Lane 9 - 12: Conventional RT with dT₂₀ at 55°C
Lane 13 - 16: Conventional RT with dN₁₂ at 55°C
Lane 17 - 20: Cyclic RT with dT₂₀ by 55°C
Lane 21 - 24: Cyclic RT with dN₁₂ at 55°C

Figure 2. Coxsackie virus A9 (AJ295200) 5’-UTR region amplification with different reverse transcription products. Coxsackie virus RNA was reverse transcribed with different reverse transcription products and then amplified with PCR with AccuPower PCR PreMix. All materials were used in accordance with manufacturer’s instructions.

M: 100 bp DNA Ladder (D-1030)
Lane 1: Reverse transcription with AccuPower RT PreMix and oligo dT primer
Lane 2: Reverse transcription with AccuPower RT PreMix and random primer
Lane 3 – 4, 7 – 8, 11 – 12: Reverse transcription with AccuPower CycleScript RT PreMix and dT₂₀
Lane 5 – 6, 9 – 10, 13 – 14: Reverse transcription with AccuPower CycleScript RT PreMix and dN₁₂
Lane 15 – 16: Reverse transcription with RT product from company C and oligo dT primer
Lane 17 – 18: Reverse transcription with RT product from company C and random primer
Lane 19 – 20: Reverse transcription with RT product from company I and oligo dT primer
Lane 21 – 22: Reverse transcription with RT product from company I and random primer

Figure 3. Long transcript production comparison. Reverse transcription was carried out with various reverse transcription products prior to PCR with AccuPower PCR PreMix.

M: 100 bp DNA Ladder (D-1030)
Gentaur: Gentaur / 1 hour incubation at 55°C
C: Company C / 1 hour incubation at 42°C
I: Company I / 1 hour incubation at 45°C
Lane 1, 3, 5: Product reverse transcribed with oligo dT primer
Lane 2, 4, 6: Product reverse transcribed with random primer

Figure 4. Transferrin receptor gene amplification with various reverse transcription products. Reverse transcription was carried out with four different reverse transcription products according to manufacturer’s instructions and then cDNA was amplified with AccuPower PCR PreMix.

M: 1 kb DNA Ladder (D-1040)
Lane 1 – 5: Reverse transcribed with AccuPower CycleScript RT PreMix (dT₂₀) / 1 hour at 55°C
Lane 6 – 10: Reverse transcribed with AccuPower CycleScript RT PreMix (dN₁₂) / 1 hour at 55°C
Lane 11 – 15: Reverse transcribed with Company C product and oligo dT primer / 1 hour at 42°C
Lane 16 – 20: Reverse transcribed with Company C product and random primer / 1 hour at 42°C
Lane 21 – 25: Reverse transcribed with Company I product and dT primer / 1 hour at 45°C
Lane 26 – 30: Reverse transcribed with Company I product and random primer / 1 hour at 45°C

Figure 5. Reaction temperatures and time comparison. Data A and B demonstrate high thermal stability of AccuPowerCycleScript RT PreMix.

A: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dT₂₀
B: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dN₁₂
Conventional RT #1: 1 hour incubation at 42°C
Cyclic RT #1: Repeat 12 times of 2 minutes at 37°C and 3 minutes at 50°C
Conventional RT #2: 1 hour incubation at 55°C
Cyclic RT #2: Repeat 12 times of 1 minutes at 37°C, 3 minutes at 47°C, and 3 minutes at 55°C
M: 100 bp DNA Ladder (D-1030)
Lane 1, 5, 9, 13, 17, 21, 25, 29: 100 ng of HeLa cell total RNA used for reaction
Lane 2, 6, 10, 14, 18, 22, 26, 20: 10 ng of HeLa cell total RNA used for reaction
Lane 3, 7, 11, 15, 19, 23, 27, 31: 1 ng of HeLa cell total RNA used for reaction
Lane 4, 8, 12, 16, 20, 24, 28, 32: 100 pg of HeLa cell total RNA used for reaction

Figure 6. Data C and D demonstrate superior productivity of AccuPower CycleScript RT PreMix..

C: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dT₂₀
D: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dN₁₂
10 min: 2 times of 2 minutes at 37°C and 3 minutes at 50°C
20 min: 4 times of 2 minutes at 37°C and 3 minutes at 50°C
60 min: 12 times of 2 minutes at 37°C and 3 minutes at 50°C
M: 100 bp DNA Ladder (D-1030)
Lane 1, 5, 9, 13, 17, 21: 100 ng of HeLa cell total RNA used for reaction
Lane 2, 6, 10, 14, 18, 22: 10 ng of HeLa cell total RNA used for reaction
Lane 3, 7, 11, 15, 19, 2: 1 ng of HeLa cell total RNA used for reaction
Lane 4, 8, 12, 16, 20, 24, 28, 32: 100 pg of HeLa cell total RNA used for reaction