Specifications

 

 

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. Performance of the AccuPower^® RocketScript™ RT PreMix, RNase H Minus in two-step RT-PCR.
Target mRNAs ranged from 500 bp to 11 kb were reverse transcribed by using AccuPower^® RocketScript™ RT PreMix, RNase H Minus. With each cDNA, PCR was performed by using AccuPower^® ProFi Taq PCR PreMix (Cat. no. K-2631 from Bioneer) Template sizes are indicated above the gel image.

M1: 1 kb DNA Ladder (Cat. no. D-1040 from Bioneer)
M2: Lambda DNA/Hind III Markers (Cat. no. D-1050 from Bioneer)

 

 

 

 

Figure 2. Comparison of cDNA synthesis efficiency of AccuPower^® RocketScript™ RT PreMix, RNase H Minus and other suppliers’ products.

Synthesis of cDNA was conducted at 50℃ or 42℃ for 10, 20, 30 and 60 min with 1 µg of 3.4 kb to 9 kb RNA transcript
(total RNA from HeLa cells) as a template by using AccuPower^® RocketScript™ RT PreMix, RNase H Minus and other kits from competitors. With each cDNA, PCR was performed by using AccuPower^® ProFi Taq PCR PreMix (Cat. no. K-2631 from Bioneer). PCR products were analyzed on a 1% alkaline agarose gel.

Lane 1: RT 10 min Lane 2: RT 20 min
Lane 3: RT 30 min Lane 4: RT 60 min
M: 1 kb DNA Ladder (Cat. no. D-1040 from Bioneer)

 

 

 

 

Figure 3. Thermal stability of AccuPower^® RocketScript™ RT PreMix, RNase H Minus.

Reverse Transcription using AccuPower^® RocketScript™ RT PreMix, RNase H Minus was performed at 50℃ to 70℃ for 30 min with 100 ng, 10 ng, 1 ng and 100 pg of 600 bp RNA transcript as a template. Even at 70℃, AccuPower^® RocketScript™ RT PreMix, RNase H Minus successfully completed cDNA synthesis. With each cDNA, PCR was performed by using AccuPower^® PyroHotStart Taq PCR PreMix (Cat. no. K-2611 from Bioneer)

Lane 1: 100 ng of total RNA from HeLa cells
Lane 2: 10 ng of total RNA from HeLa cells
Lane 3: 1 ng of total RNA from HeLa cells
Lane 4: 100 pg of total RNA from HeLa cells
M: 100 bp DNA Ladder (Cat. no. D-1030 from Bioneer)

 

 

 

Figure 4. Sensitivity comparison of RTase products with various targets.
Various target mRNAs were reverse transcribed at 50℃ for 30 min by using AccuPower^® RocketScript™ RT PreMix, RNase H Minus and competitor’s product, respectively. With each cDNA, PCR was performed by using AccuPower^® PyroHotStart Taq PCR PreMix (Cat. no. K-2611 from Bioneer)

Lane 1: 10 ng of total RNA from HeLa cells
Lane 2: 1 ng of total RNA from HeLa cells
Lane 3: 100 pg of total RNA from HeLa cells
Lane 4: 10 pg of total RNA from HeLa cells
M: 100 bp DNA Ladder (Cat. no. D-1030 from Bioneer)

 

 

 

Figure 5. Broad dynamic range of AccuPower^® RocketScript™ RT PreMix, RNase H Minus.
First-strand cDNA was synthesized using the AccuPower^® RocketScript™ RT PreMix, RNase H Minus. cDNA was amplified using the AccuPower^® Plus DualStar™ qPCR PreMix (Cat. no. K-6600 from Bioneer) on the Exicycler™ 96 Real-Time Quantitative Thermal Block from Bioneer. The standard curve illustrates high linearity (R2 = 0.999) across a broad range of input RNA, suggesting that the relative representation of specific RNA transcripts is preserved in the cDNA pool regardless of the abundance of total RNA. Amplification was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 1 pg) and mouse total RNA (10 μg to 1 pg).