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    Friday, 01 March 2013 13:18

    AccuPower RocketScript Cycle RT PreMix

    Rate this item
    (1 Vote)

    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.

    accupower-antibodies-gentaur-aproketscript overview 

    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

    accupower-antibodies-gentaur-aproketscript-cycle figure1

    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

    accupower-antibodies-gentaur-aproketscript-cycle figure2

    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

     

     accupower-antibodies-gentaur-aproketscript-cycle figure3

    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)

     accupower-antibodies-gentaur-aproketscript-cycle figure4

    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

    accupower-antibodies-gentaur-aproketscript-cycle figure5

    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

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