Products | Genomics

 

PrimeScript™ – Reverse Transcriptase of a New Generation

  
  MoBiTec   PrimeScript™ is an RTase that Takara Bio originally developed from M-MLV RTase. This enzyme carries excellent extension faculty and offers robust reverse transcription of up to 12 kb mRNA with high sensitivity and large yield. Also, it is able to reverse transcribe even complex highly structured RNA at usual reaction temperature (42 °C).

   TaKaRa  
     
Content
Overview
Comparison of RTases
PrimeScript™ 1st strand cDNA Synthesis Kit
PrimeScript™ RT-PCR Kit - Two step RT-PCR
PrimeScript™ One Step RT-PCR Kit
High Fidelity PrimeScript™ RT-PCR Kit
PrimeScript™ qRT-PCR Kits - For Fast and Accurate Quantification
Order Information
Literature
 

Features:

  • Extention of longer templates due to strong displacement extension activity

  • Unsurpassed reverse transcription efficiency even on highly structured RNA

  • Highly accurate cDNA synthesis at 42 °C with minimal background
      
 

 

 

Why PrimeScript™ RTase is outstanding?

The main cause of interference of cDNA synthesis by reverse transcription, using complex structure RNA or long chain RNA as template, is non-specific annealing of RTase itself to high order structures of RNA. Also, non specific product derived from mis-priming of RTase has negative effect on RT efficiency or full length cDNA synthesis. To minimize the above factors, high temperature reaction is frequently used to relax secondary structures, which in general is not ideal. PrimeScript™ RTase is an enzyme that lowers inefficient cDNA synthesis and mis-priming by controlling non-specific annealing and preventing primer-dimer. As it contains strong strand displacement activity, this enzyme is capable of synthesizing cDNA with insignificant background for complex structure RNA or long chain RNA at 42 °C, which is known to be less damaging.

 

      
 
             
 
 

A comparison of PrimeScript™ RTase and three competitor RTases respective elongation, sensitivity and yield

After reverse transcription of total RNA from human cell culture or tissue using manufacturers' recommended protocols, PCR was done under the same conditions.

As a result, PrimeScript™ RTase achieved transcription of up to 12 kb mRNA with excellent elongation. When using 10 ng of template RNA, PrimeScript™ RTase showed excellent cDNA yield and higher sensitivity than any other of the competitors' RTases:

     
                     
 
A comparison of PrimeScript™ RTase and three competitor RTases respective elongation, sensitivity and yield
Target gene: transferrin receptor gene (TFR); Template: total RNA prepared from HL60 cell lines   Target gene: dystrophin gene; Template: total RNA prepared from Human heart  
  Lane / Amount of template: Lane 1: 0 ng; 2: 1 ng; 3: 10 ng; 4: 100 ng; 5: 1000 ng
M: (left) pHY Marker; (right) Lambda-Hind III digest 		 
 
       

 

 

            
  top

  
     

PrimeScript™ 1st strand cDNA Synthesis Kit

PrimeScript™ 1st strand cDNA Synthesis Kit contains all the reagents necessary to synthesize 1st strand cDNA from total or poly(A)+ RNA using PrimeScript™ RTase. 1st strand cDNAs synthesized with this kit can be used for a variety of applications including 2nd strand synthesis, hybridization, PCR amplification, and real-time PCR.

Order Information
order # description   amount
2680A-TK PrimeScript™ Reverse Transcriptase (200 U/µl) 10'000 U
6110A-TK PrimeScript™ 1st strand cDNA Synthesis Kit; Components:
PrimeScript™ RTase (200 U/µl)
5 x PrimeScript™ Buffer
RNase Inhibitor (40 U/µl)
dNTP Mixture (10 mM each)
Oligo dT Primer (50 µM)
Random 6 mers (50 µM)
RNase free H2O

10'000 U
200 µl
1000 U
50 µl
50 µl
100 µl
1 ml 		50 react. 
     
             
     

PrimeScript™ RTase is included in TaKaRa's one- or two-step PrimeScript™ RT-PCR Kit. These kits offer PrimeScript™ RTase's robust reverse transcription as well as highly efficient amplification thanks to TaKaRa Ex Taq™ Hot Start DNA Polymerase. The combination of TaKaRa Bio's new PrimeScript™ RTase with TaKaRa Ex Taq™ HS Polymerase results in reliable, efficient, and reproducible RT-PCR amplification from a large variety of RNA templates.

 

      
                     
     

PrimeScript™ RT-PCR Kit — Two step RT-PCR

     
      Reverse transcription and subsequent amplification were performed using PrimeScript™ RT-PCR Kit and RT-PCR Kit using AMV RTase. Targets of various lengths of human heart total RNA (Dystrophin) or HL60 cell total RNA (TFR) were templates. Excellent extension and amplification were confirmed on targets from 2.2 kbp to 12 kbp in length using the PrimeScript™ RT-PCR Kit.  
PrimeScript™ RT-PCR Kit — Two step RT-PCR
  Template gene: TFR gene; Template: 0;1;10;100;1000 ng   Target gene: dystrophin gene; Template: 0;1;10;100;1000 ng
 
     
Order Information
order # description   amount
RR014A-TK PrimeScript™ RT-PCR Kit; Components:
PrimeScript™ RTase (for 2 step)
5 x PrimeScript™ Buffer
RNase Inhibitor
dNTP Mixture (10 mM each)
Oligo dT Primer (2.5 µM)
Random 6 mers (20 µM)
TaKaRa Ex Taq™ HS (5 U/µl)
10 x PCR buffer II
Control F-1 primer** (20 µM)
Control R-1 primer*** (20 µM)
Positive control RNA (2 x 105 copies/µl)
RNase free H2O
25 µl
5 x 200 µl
1000 U
150 µl
50 µl
50 µl
125 U
10 x 250 µl
10 µl
10 µl
20 µl
1 ml		 50 react.* 
*50 reactions of [Reverse transcription 20 µl —› PCR 50 µl]
** Upstream sense primer for positive control RNA
*** Downstream anti-sense primer for positive control RNA
     
     

 

top

      
     

PrimeScript™ One Step RT-PCR Kit

     
      A comparison of PrimeScript™ One Step RT-PCR and three competitor RTases
Reverse transcription and subsequent amplification were performed using PrimeScript™ One Step RT-PCR Kit. Targets of various lengths of human heart total RNA (Dystrophin) or HL60 cell total RNA (TFR) were templates. Company A,C,D were tested using the manufactures' recommended protocols. Excellent extension and amplification were confirmed on targets from 0.5 kbp to 8 kbp in length using the PrimeScript™ One Step RT-PCR Kit.

     
     
PrimeScript™ One Step RT-PCR Kit
Template: human total RNA
Lane: 1:TFR 0.5 kb; 2: CCND 2.1 kb; 3: CCND 2.8 kb; 4: TFR 4.4 kb; 5:Dystrophin 6kb; 6: Dystrophin 8 kb; M: lambda-Hind III digest
     
     
Order Information
order # description   amount
RR018A-TK PrimeScript™ One Step RT-PCR Kit; Components:
PrimeScript™ RTase (for 1 step)
RNase Inhibitor (40 U/µl)
TaKaRa Ex Taq™ HS (5 U/µl)
RNase free H2O
dNTP Mixture (10 µM each)
10 x One Step RT-PCR Buffer
One Step Enhancer Solution
Control F-1 primer** (20 µM)
Control R-1 primer*** (20 µM)
Positive control RNA (2 x 105 copies/µl)
25 µl
2000 U
250 U
2 x 1.1 ml
100 µl
250 µl
50 µl
20 µl
20 µl
20 µl 		50 react.* 
*50 reactions of [Reverse transcription 20 µl —› PCR 50 µl]
** Upstream sense primer for positive control RNA
*** Downstream anti-sense primer for positive control RNA
     
     

 

 

              
     

High Fidelity PrimeScript™ RT-PCR Kit

     
      High Fidelity PrimeScript™ RT-PCR Kit contains PrimeScript™ RTase along with the high fidelity DNA polymerase PrimeSTAR® HS. Combining Takara Bio's new PrimeScript™ RTase with PrimeSTAR® HS DNA polymerase results in great accuracy, reliable, efficient, and reproducible RT-PCR amplification from a variety of RNA templates.

     
     
Accuracy of various RTases
RTase Bases sequenced Error (bases) Error rate (%)
PrimeScript™ 201'297 7 0.0035
Company A RTase 2 166'227 9 0.0054
Company A RTase 161'409 11 0.0068
Company C RTase 132'962 13 0.0098
Control (PCR only) 156'188 1 0.0006
Accuracy of various RTases
 
     
Order Information
order # description   amount
R027A-TK High Fidelity PrimeScript™ RT-PCR Kit; Components:
PrimeScript™ RTase (for 2 step)
5 x PrimeScript™ Buffer
RNase Inhibitor (40 U/µl)
dNTP Mixture (10 mM each)
Oligo dT Primer (2.5 µM)
Random 6 mers (20 µM)
PrimeSTAR® HS DNA Polymerase
5 x PrimeSTAR® P CR buffer
Control F-1 primer** (20 µM)
Control R-1 primer*** (20 µM)
Positive control RNA (2 x 105 copies/µl)
RNase free H2O
25 µl
200 µl
1000 U
50 µl
50 µl
50 µl
25 µl
500 µl
10 µl
10 µl
20 µl
550 µl		 50 react.* 
*50 reactions of [Reverse transcription 20 µl —› PCR 50 µl]
** Upstream sense primer for positive control RNA
*** Downstream anti-sense primer for positive control RNA

top


      
     

PrimeScript™ qRT-PCR Kits - For Fast and Accurate Quantification

One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time)

One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) is designed for real time one step RT-PCR using SYBR® Green I1,3 for detection. In this kit, RT-PCR can be performed all in a single tube subsequently, therefore the operation is simple, and the risk of contamination is minimized. As amplified products are monitored in real time, there is no need to verify them through electrophoresis after PCR. This kit is suitable for detection of tiny amounts of RNA like RNA virus.
This kit uses PrimeScript™ RTase, which has excellent extendibility and can efficiently synthesize cDNA in short time periods, and TaKaRa Ex Taq™ HS, high efficiency hot start PCR enzyme, both are optimized for one step RT-PCR.

 

One Step PrimeScript™ RT-PCR Kit (Perfect Real Time)

One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) is designed for real time one step RT-PCR using TaqMan® probe2,3 for detection. In this kit, RT-PCR can be performed all in a single tube subsequently, therefore the operation is simple, and it minimizes the risk of contamination. Also, amplified products are monitored in real time, so there is no need to verify them through electrophoresis after PCR. This kit is suitable for detection of tiny amounts of RNA like RNA virus.
This kit uses PrimeScript™ RTase, which has excellent extendibility and can efficiently synthesize cDNA in short time periods, and TaKaRa Ex Taq™ HS, high efficiency hot start PCR enzyme, and both are optimized for one step RT-PCR.
1 Includes dNTP Mixture, Mg2+ and SYBR® Green I
2 Includes dNTP Mixture and Mg2+.
3 ROX™ Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM® 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX™ Reference Dye (50×) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX™ Reference Dye II is recommended. The use of ROX™ Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice® Real Time System, Smart Cycler® or LightCycler® real time instruments.

Applicable real time PCR instruments

  • Thermal Cycler Dice™ Real Time System (TaKaRa Cat.# TP800-TK)
  • ABI PRISM® 7000/7700/7900HT, 7300/7500 Real-Time PCR System (Applied Biosystems)
  • LightCycler® (Roche Diagnostics)
  • Smart Cycler® System*3/Smart Cycler® II System*3 (Cepheid),
  • and other
      
             
 
PrimeScript Amplification curve   Primescript melting curve
Primescript 2nd derivate   Primescript standard curve
Detection of Rat Rplp2 (ribosomal protein, lange P2)
Using total RNA 6.4 pg-100 ng that was prepared from rat liver and sterilized water (negative control) as a template, Real-Time One Step RT- PCR was performed with Thermal Cycler Dice™ Real Time System. After reaction, the amplification curves and the melting curves were confirmed, and the standard curve was created. Target gene was detected in the range of 6.4 pg-100 ng of total RNA. The melting curves show that the same amplification products were obtained in all the reactions. The linearity of standard curve was obtained within the wide range of template.
 
     
Order Information
order # description   amount
RR064A-TK One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) Components:
2× One Step RT-PCR Buffer III1
TaKaRa Ex Taq™ HS (5U / µl)
PrimeScript™ RT enzyme Mix II
RNase Free dH2O
ROX Reference Dye (50×conc.)3
ROX Reference Dye II (50×conc.)3

  3x 840 µl
100 µl
100 µl
2 x 1.25 ml
  100 µl
  100 µl
 100 react.
RR066A-TK One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) Components:
2× One Step SYBR® RT-PCR Buffer III2
TaKaRa Ex Taq™ HS (5U / µl)
PrimeScript™ RT enzyme Mix II
RNase Free dH2O
ROX Reference Dye (50×conc.)3
ROX Reference Dye II (50×conc.)3

  3 x 840 µl
100 µl
100 µl
2 x 1.25 ml
  100 µl
  100 µl 		100 react.
1 Includes dNTP Mixture and Mg2+.
3 Includes dNTP Mixture, Mg2+ and SYBR® Green I
2 ROX™ Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM® 7000/7700/7900HT and Applied Biosystems 7300 Real-Time PCR System, the use of ROX™ Reference Dye (50×) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX™ Reference Dye II is recommended. The use of ROX™ Reference Dye or Dye II is optional. It is not required for use with Thermal Cycler Dice™ Real Time System, Smart Cycler® or LightCycler® real time instruments.


     
      top

      
     

Order Information

Content
Overview
Comparison of RTases
PrimeScript™ 1st strand cDNA Synthesis Kit
PrimeScript™ RT-PCR Kit - Two step RT-PCR
PrimeScript™ One Step RT-PCR Kit
High Fidelity PrimeScript™ RT-PCR Kit
PrimeScript™ qRT-PCR Kits - For Fast and Accurate Quantification
Order Information
Literature

Please click on the respective order # for further details.

     
     
List of all PrimeScript™ products
order # product amount
2680A-TK PrimeScript™ Reverse Transkriptase 10'000 U
2680B-TK PrimeScript™ Reverse Transkriptase (2680A-TK x 4) 40'000 U
2680C-TK PrimeScript™ Reverse Transkriptase (2680A-TK x 10) 100'000 U
2680Q-TK PrimeScript™ Reverse Transkriptase (Sample size) 2'000 U
6110A-TK PrimeScript™ 1st strand cDNA synthesis kit 50 react.
6110B-TK PrimeScript™ 1st strand cDNA synthesis kit (6110A-TK x 4) 200 react.
R027A-TK PrimeScript™ High Fidelity RT-PCR Kit 50 react.
R027B-TK PrimeScript™ High Fidelity RT-PCR Kit (R027A-TK x 4) 200 react.
RR064A-TK One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) 100 react.
RR064B-TK One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (RR064A-TK x 5) 500 react.
RR066A-TK One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) 100 react.
RR066B-TK One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) (RR066A-TK x 5) 500 react.
RR014A-TK PrimeScript™ RT-PCR Kit 50 react.
RR014B-TK PrimeScript™ RT-PCR Kit (RR014A-TK x 4) 200 react.
RR018A-TK PrimeScript™ One Step RT-PCR Kit 50 react.
RR018B-TK PrimeScript™ One Step RT-PCR Kit (RR018STK x 4) 200 react.
RR037A-TK PrimeScript™ RT Reagent Kit (Perfect Real Time) 200 react.
store at -20°C
     
             
      top        
     

 

Literature

Brochures

Data sheets

      
      top
      
     
<< PrimeSTAR™ HS DNA Polymerase Mastermix 16S - No False-Positive qPCR Results Anymore >>
     
                     

| home | search | site map | order | Impressum |

©2007 MoBiTec GmbH • All rights reserved • www.mobitec.com