Takara-PrimeScriptTM RT reagent Kit
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, Table of contents
I. Kit Components (2)
II. Storage (2)
III. Principle (2)
IV. Protocol (3)
V. Protocol: reverse transcription (3)
VI. Protocol: Real-time PCR (4)
VII. Appendix (6)
VIII. Related Products (9)
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, I. Kit Components:PrimeScript TM RT reagent Kit is designed to perform the reverse transcription opti-
mized for real-time RT-PCR. It uses PrimeScript TM RTase, which features excellent
extendibility and makes fast, efficient cDNA template synthesis for Real Time PCR
possible. The step experimental procedure is simple and suitable for high through-
put analysis. This kit can be used in combination with Real Time PCR reagent,
SYBR ? Premix Ex Taq TM (Perfect Real Time) or Premix Ex Taq TM (Perfect Real
Time) for 2 step Real Time RT-PCR.
. 5 X PrimeScript TM Buffer (for Real Time) *
400 μl 2. PrimeScript TM RT Enzyme Mix I
100 μl 3. Oligo dT Primer
50 μM 100 μl 4. Random 6 mers
100 μM 100 μl 5. RNase Free dH 2O
ml 6. EASY Dilution Buffer (for Real Time PCR) *2
ml
*1: contains dNTP Mixture and Mg 2+.
*2: For measured dilution of total RNA or cDNA used as diluted solution.
In contrast to dilution with water or TE, EASY Dilution Solution facilities accurate
low concentration dilution. Diluted template reagent can be applied as the
template for reverse transcription or PCR reactions, because Easy Dilution
Solution does not inhibit either reverse transcription or PCR enzyme activity.
EASY Dilution Solution is also available separately.
EASY Dilution (for Real Time PCR) (TaKaRa Cat.#9160)
Note: EASY Dilution has been tested with TaKaRa’s Bio real-time PCR reagent.
Compatibility with products from other manufacturers has not yet been verified.
Reagents and instruments not supplied in this kit
. Thermal Cycler (or 37°C, 42°C Water Bath, 85°C heat block)
2. 0.2ml and .5ml micro tube (for reverse transcription)
3. Micropipettes and pipette tips (autoclaved)
-20℃(1) Makes fast, efficient synthesis of cDNA templates for Real Time PCR possible.
This kit is best suited for two step real-time RT-PCR.
(2) The kit includes Random 6 mers and Oligo dT Primer for use as reverse
transcription primers. The reaction can be performed using mixture these two
primers, or the primer can be selected based on the purpose of the experiment.
Furthermore, Gene Specific Primers can be used for specific gene detection.
(3) A standard curve must be generated for the quantitation of Real-Time RT-PCR.
Dilution of total RNA or cDNA after reverse transcription is necessary because
low concentrations are required for a viable standard curve. However, dilution
with water or TE can narrow the range of the curve due to unstable dilution at
low concentrations. Using EASY Dilution Solution (for Real Time PCR), for
dilution causes the results to be accurate at lower concentrations and facilitates
creation of a wide-range standard curve. II. Storage:
III. Principle:
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, IV. Protocol:Following is the protocol for this kit. Please read it carefully before you use.
(1) It is convenient to prepare a master mix of reagents containing (RNase Free
dH 2O, buffer, enzymes, etc). Using such a mixture allows accurate dispensing of
reagents, minimizes pipetting losses, and avoids repeated dispensing of each
reagent. This helps to minimize experimental variability.
(2) Gently spin down the PrimeScript TM RT Enzyme Mix I prior to pipetting. Pipet
enzymes slowly and carefully because of the viscosity of the 50% glycerol in this
solution.
(3) Use new disposable pipette tips to avoid contamination between samples when
transferring reagents.
(Refer VII. B. RNA sample preparation (page 8) for RNA preparation)
. Prepare the following reaction mixture on ice.
Prepare a slightly larger amount of master mix than is required to compensate for
pipetting losses. After dispensing aliquots of this mix into the micro tubes, add the
RNA sample.
< For 1 reaction >
Reagent
Amount Final concentration 5 × PrimeScript TM Buffer (for Real Time)
2 μl 1 ×PrimeScript TM RT Enzyme Mix I
0.5 μl Oligo dT Primer (50 μ M)*
0.5 μ l 25 pmol Random 6 mers (100 μ M)*
0.5 μ l 50 pmol total RNA
RNase Free dH 2O
Total 10 μ l *2
*1. Using both Oligo dT Primer and Random 6 mers, efficient synthesis of cDNA
from total mRNA can be accomplished. The required amount of primer for
exclusive use of each primer or a Gene Specific Primer is as follows.
Primer Amount Total Amount (pmol)
Oligo dT Primers (50 μM) 0.5 μl 25 pmol
Random 6 mers (100 μ M) 0.5 μl 50 pmol
Gene Specific Primer (2 μM) 0.5 μl 1 pmol
*2: It is possible to scale up the RT reaction as needed. Up to 500 ng of total RNA can be reverse transcribed in 10 μl of the reaction mixture.
2. Incubate the reaction mixture under the following condition.
37℃, 5 minutes*3 (Reverse transcription)
85℃, 5 sec (Inactivation of reverse transcriptase with heat treatment)
4℃V. Protocol: reverse transcription:
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*3. When using a Gene Specific Primer:
Perform the reverse transcription at 42℃ for 5 minutes.
If non-specific amplification products are observed at the PCR step,
resetting this temperature to 50℃ may improve the results.
Note: The volume of the reaction mixture obtained in step 2 must be less
than 0% of the total PCR reaction volume for real-time PCR.The following protocol is for real-time PCR using SYBR ? Premix Ex Taq TM (Perfect
Real Time) (TaKaRa Cat.#RR04 A) with templates generated with this kit. If per-
forming real-time PCR with TaqMan probe detection, TaKaRa recommends use of
Premix Ex Taq TM (Perfect RealTime) (TaKaRa Cat.#RR039A)
* Follow the instrument manual recommended conditions (Applied Biosystems).
. Prepare the PCR reaction described below.
< Per reaction >
Reagent Amount Amount
Final Concentration SYB R ? Premix Ex Taq TM (2 X) 10 μl 25 μl
1 X PCR Forward Primer (10 μM) 0.4 μl 1 μl
0.2 μM* PCR Reverse Primer (10 μM) 0.4 μl 1 μl
0.2μM* ROX Reference Dye or Dye II (50X)*3 0.4 μl 1 μl
1 X RT reaction solution (cDNA solution)
2 μl 4 μl
*2dH 2O (satirized distilled water) 6.8 μl 18 μl
Total 20 μl *4 50 μl *4
*1. The final concentration of primers can be 0.2 μM in most reactions. When it does
not work, determine the optimal concentrations within the range of 0.1 - 1.0 μM.
*2. It is recommended to apply DNA template in less than 100 ng per 20 μl of re
action mixture. When the RT reactant (cDNA) is used as a template, it should be
added in less than 0% volume of PCR reaction mixture.
*3. The dye concentration of ROX Reference Dye II (50X) is lower than ROX
Reference Dye (50X). Use of ROX Reference Dye II (50X) is recommended
for analysis using the 7500 Real-Time PCR System or 7500 Fast Real-Time
PCR System. This solution gives higher signal value after correction than
ROX Reference Dye (50x) apparently, but the analysis result is equal.
Use ROX Reference Dye (50X) for the ABI PRISM 7000/7700/7900HT and 7300
Real-Time PCR System.
*4. The reaction of 50 μl is for 96-well plate, singletube and 8-strip tube.
The reaction of 20 μl is for 384-well plate.
VI. Protocol: Real-time PCR < Protocol for using ABI PRISM 7000 / 7700 / 7900HT and 7300 / 7500 / 7500 Fast Real-Time PCR System >
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, Shuttle PCR standard protocol
Stage : Early denaturing
Reps:
95℃ 0 sec.
Stage 2: PCR reaction
Reps: 40
95℃5 sec.
60℃30 sec.~ 34 sec.*
Dissociation Stage
*Set at 30 sec. for 7700 and 7900HT,
3 sec. for 7000 and 7300, 3
4 sec.
for 7500.
Stage : Initial denaturation step
Reps:
95℃ 0 sec.
Stage 2: PCR reaction
Reps: 40
95℃3 sec.
60℃25 sec.
Dissociation Stage
Note: This product combines the high performance of TaKaRa Ex Taq TM HS, which
is an enzyme for hot start PCR utilizing Taq antibody. Initial denaturation step prior
to PCR should be at 95℃ for 10 sec. No need to heat at 95℃ for (5-) 5 min. as the
initial denaturation, required for chemically modified Taq polymerase. If longer heat
treatment is provided, the enzyme activity descreases and the amplification efficien -
cy and the accuracy in quantification can also be affected.
3. Reaction Analysis
After completing reaction, verify amplification curve and dissociation curve, create
standard curve if quantitative analysis is necessary. *
*Refer manual of Real Time PCR instrument for detail analysis.
< ABI PRISM 7000 / 7700 / 7900HT, 7300 / 7500 Real-Time PCR System >
< 7500 Fast Real-Time PCR System >
2. Start Reaction
Shuttle PCR standard protocol (below) is recommended. Try this protocol firstly,
and optimize the reaction condition if needed. Try 3 step PCR protocol, when
shuttle protocol is difficult (ex. Tm value of primers is low.)
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, VII. Appendix: A. Experimental example: Reverse Transcription Reaction Time and Amount of
cDNA Synthesis
[ Process ]
Reverse Transcription
Reagent: PrimeScript TM RT reagent Kit (Perfect Real Time)
Template: Mouse liver total RNA (2 pg ~ 2 μg and sterilized water)
Reaction mixture: 20 μl
Primer: Random 6 mers
Reaction Condition: 37℃ 5, 30, 60 min.→ 85℃ 5 seconds → 4℃
Real Time PCR
Reagent: SYBR ? Premix Ex Taq TM (Perfect Real Time)
Template: 2 μl of reverse transcription reaction mixtures from above 20μl reactions
Reaction mixture: 25 μl
Measured Gene: Mouse Actb
Reaction Condition: Thermal Cycler Dice ? Real Time System Standard protocol
15 min. (purple) Rsq: 0.999 Eff = 98.7% Y= - 3.522 * LOG (X) + 33.94
30 min. (red) Rsq: 0.999 Eff = 93.3% Y= - 3.495 * LOG (X) + 34.61
60 min. (brown) Rsq: 0.999 Eff = 95.2 Y= - 3.441 * LOG (X) + 34.28
The reverse transcription reaction time was set at 5, 30, and 60 min. and a compari-
son was performed. In this experiment, all three reaction times showed equally efficient
reverse transcription over a wide range of the template concentration.
[ Result ]
Amplification curve
5 min. (purple)
30 min. (red)
60 min. (brown)
Standard curve
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, B. Preparation of RNA sample It is important to use a highly pure RNA samples for better cDNA yield. It is essential to inhibit RNase activity in the cells and also to prevent RNase derived from equipment and solutions used. Extra precautions should be taken during the sample preparation, including use of clean disposable gloves, dedication of a exclusively table for exclusive use for RNA preparation, and avoiding unnecessary speaking during assembly to prevent RNase contamination from operater sweat or saliva. [ Equipment ]Disposable plastic equipment should be used. For glass tools should be treated with the following procedure prior to use. (1) Hot-air sterilization (180°C, 60min), or (2) Treatment with 0.1% diethylpyrocarbonate (DEPC) at 37°C for 12 hours, followed by autoclaving at 120°C for 30 min. to remove DEPC.It is recommended that all the equipment be used exclusively for RNA preparation. [Reagent]All reagents to be used in this experiment must be prepared using tools which were treated as described in previous section (Hot-air sterilization (180°C, 60min) or DEPC treatment), and all distilled water must be treated with 0.1%DEPC and auto -claved. All reagents and distilled water should be used exclusively for RNA experi -ments.[Preparation of RNA sample]Use of highly purified RNA obtained by GTC (Guanidine thiocyanate) method, etc is recommended. RNA samples should be suspended in sterilized distilled water or TE Buffer solution prior to reverse transcription .[Contamination with Genomic DNA and its countermeasure]Some cases, a total RNA sample may contain a small amount of genomic DNA, which could potentially become a PCR template. This could result in inaccurate re-sults. To avoid this to situation, the following countermeasure is recommended: (1) design primers which will not amplify genomic DNA, or (2) remove genomic DNA by DNase I treatment (1) Designing a primer which will not amplify genomic DNA: It is possible to design a primer that will not amplify genomic origin of DNA based on the exon and intron structure of genomic DNA. First, confirm the target genomic structure, and select a large intron region. Then design primers on the upstream and downstream sides of the intron regin region. If the intron is large enough, genomic DNA amplification will not occur.
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Even if size of the intron is not large, products resulting from amplification of
genomic templates would be larger than products originating from cDNA,
allowing identification by dissociation curve analysis. However, this approach
cannot be applied for single-exon genes or genes that contain pseudogene.
Moreover, species without introns or new species without well-identified
genomic structures could demonstrate similar problems. In this case, we
recommends performing the DNase I treatment described in (2).
(2) After extract total RNA remove genomic DNA by DNase I (RNase-free) (TaKaRa
Cat.#2215A) treatment. After the reaction, DNase I should be inactivated by
either heal treatment or phenol/chloroform extraction.
Procedure
. Prepare the following reagents:
total RNA
20~50 μg 10 X DNase I Buffer 5 μl
RNase Inhibitor 20 U
DNase I (RNase-free) 2 μl (10U)
DEPC treated water to 50 μl
2. Incubate at 37℃ for 20 min.
3. Perform one of the following procedures to inactivate DNase I
A. Heat Treatment
(1) Add 2.5μl of 0.5M EDTA, incubate at 80℃ for 2 minutes
(2) Increase reaction volume to 100μl with DEPC treated water
B. Phenol/Chloroform Extraction
(1) Mix 50 μl of DEPC treated water and 100 μl of phenol/chloroform/
isoamyl alcohol (25:24: ) together.
(2) Centrifuge at 5,000 rpm for 5 minutes, at room temperature
transfer the upper layer to a new tube.
(3) Add equal amount of Chloroform/ /isoamyl alcohol (24: ) and mix.
(4) Centrifuge at 5,000 rpm for 5 minutes, at room temperature
transfer upper layer to new tube.
4. Add 10 μl of 3 M sodium acetate and 250 μl of cold ethanol, then incubate on ice
for 0 min.
5. Centrifuge at 5,000 rpm for 5 minutes at 4℃and remove the top clear liquid.
6. Wash the precipitate, with 70% ethanol and then, centrifuge at 5,000
rpm for 5 minutes at 4℃and remove the supernatant.
7. Dry the precipitate.
8. Dissolve the precipitate in the proper amount of DEPC treated water.
[ Confirmation of Genomic DNA Contamination ]
The presence of genomic DNA can be verified by Real Time PCR without reverse
transcription. It is convenient to use a primer that can do PCR amplification from
both genomic DNA and mRNA for this experiment, and one tube should include an
exogenous genomic DNA aliquot . In addition, a primer which was designed not to
amplify genomic DNA could amplify derived from pseudogene. In this case, this
procedure can also be use to verify DNA.
TAKARA BIO INC.(Perfect Real Time)
https://www.wendangku.net/doc/6615051314.html, VIII. Related Products:SYBR ? Premix Ex Taq TM (Perfect Real Time) (TaKaRa Cat.#RR04 A)
Premix Ex Taq TM (Perfect Real Time) (TaKaRa Cat.#RR039A)
EASY Dilution (for Real Time PCR) (TaKaRa Cat.#9160)
TAKARA BIO INC.(Perfect Real Time)
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