Table of content. One Step SYBR R PrimeScript TM RT-PCR Kit II (Perfect Real Time) I. Description...2. II. Principle III. Kit Contents...

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1 Table of content I. Description...2 II. Principle... 2 III. Kit Contents...4 IV. Storage...5 V. Features...5 VI. Note...5 VII. Protocol...6 VIII. Experiment Example...11 IX. Appendix...12 X. Guideline for designing of primer...13 XI. Related Products...14

2 I. Description: One Step SYBR R PrimeScript TM RT-PCR Kit II is designed for Real- Time One Step RT-PCR including SYBR R Green I* 1, * 2. 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 monitoring in real time, so there is no need to verify them through electrophoresis after PCR. This kit is suitable for detection of tiny amount of RNA like RNA virus. This kit uses PrimeScript TM RTase, which has excellent extendibility and can efficiently synthesize cdna in short time period, and TaKaRa Ex Taq TM HS, high efficiency hot start PCR enzyme, and they are optimized for one step RT-PCR. Combining the TaKaRa Bio RT-PCR technology with these enzymes allow this kit to have a more efficient gain of RT-PCR product. Applicable real time PCR instruments; * 3 ABI PRISM R 7000/7700, 7300/7500 Real-Time PCR System (Applied Biosystems) LightCycler R (Roche Diagnostics), and other Thermal Cycler Dice TM Real Time System (TaKaRa Cat.#TP800) II. Principle: *1. SYBR R Green I is licensed by Molecular Probes Inc. for research reagents. SYBR R is a trade mark of Molecular Probes Inc. (U.S. and Europe). *2. It is recommended to use One Step PrimeScript RT-PCR Kit for Real-Time One Step RT-PCR used with probes such as TaqMan R probe. (TaqMan R is trademark of Roche Molecular Systems. The 5' nuclease process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hofmann- La Roche Ltd. Purchase of the product does not provide a license to use this patentedtechnology.) *3. it is recommended to use One Step SYBR R PrimeScript TM RT-PCR Kit (TaKaRa Cat.#RR066A) for analyzing with Smart Cycler R System/ Smart Cycler R Ⅱ System(Cepheid) (Smart Cycler R is a trade mark of Cepheid). One Step SYBR R PrimeScript TM RT-PCR Kit perform cdna synthesis from RNA using reverse transcriptase, PrimeScript TM RTase, and PCR amplification by TaKaRa Ex Taq TM HS within one tube continuously. PCR amplification products are monitored real time by SYBR R Green I. 1. PCR PCR is a technique that amplifies only targeted region of gene from small amount of DNA. One cycle of PCR includes Heat denaturation of DNA, annealing of primer, and extension with DNA polymerase. By repeating this process, PCR allows amplification of targeted gene fragment in million times in short time period. Use of Hot Start PCR enzyme, TaKaRa Ex Taq TM HS, for amplification made this kit possible to avoid miss-priming prior to the reaction and non-specific amplification caused by primer dimer, and this kit is possible to do high quality detection. 2

3 2. RT-PCR Although RNA would not be a direct template of PCR, PCR can be applied for RNA analysis when cdna were synthesized from RNA with reverse transcriptase. This is RT-PCR and is a high quality detection of RNA. This kit uses One Step RT-PCR. The principle of this is shown in below. For One Step RT-PCR, use Specific Primer (Reverse) for PCR in reverse transcription. Then, do PCR amplification using Specific primer(forward, Reverse) and the synthesized cdna as a template. (Random Primer and Oligo dt Primer cannot be used for reverse transcription.) mrna 3 5 Specific Primer(Reverse) Synthesis of 1st strand cdna with PrimeScript TM RTase cdna 3 5 Specific Primer(Forward) 5 Synthesis of 2nd strand cdna with TaKaRa Ex Taq TM HS Amplify cdna Specific Primer(Reverse) Principle of One Step RT-PCR 3

4 3. Fluorescent spectrophotometer method [Intercalator method] This method detect fluorescence which produced in the amplification process by adding reactants (for example Intercalater: SYBR R Green I) that fluoresces by bonding to double strand of DNA to the reactive system. It fluoresces when intercalater bound with double stranded DNA that is synthesized by polymerase reaction. From detecting this fluorescence, not only quantity of amplified DNA, but also, the melting point could be measured. 1) Heat denaturation Primer Intercalater (Fluorescent substance) 2) Primer annealing Polymerase 3) Extension III. Kit Contents (for 100 reactions; 50 μ l reaction system): 1. 2X One Step SYBR R RT-PCR Buffer IV* μ l X 3 2. PrimeScript TM 1 step Enzyme Mix II* μ l 3. RNase Free dh2o.25 ml X 2 4. ROX Reference Dye (50 conc.)* μ l 5. ROX Reference Dye II (50 conc.)* μ l *1. Includes dntp Mixture, Mg 2+ and SYBR R Green I *2. Includes PrimeScript RTase, RNase Inhibitor, TaKaRa Ex Taq TM HS *3. ROX TM Reference Dye/Dye II is used for normalization of intensity by background subtraction. For ABI PRISM R 7000/7700 and Applied Biosystems 7300 Real Time PCR System, the use of ROX TM Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real Time PCR System, the use of ROX TM Reference Dye II is recommended. The use of ROX TM Reference Dye or Dye II is optional. It is not required for use with Smart Cycler R or LightCycler R real time instruments. Reagents or equipment not included in the kit. 1. Gene amplification system for Real-Time PCR 2. Reaction tube or plate exclusive for the real time PCR 3. PCR Primer * 1 4. Micropipetts and pipette tips (autoclaved) *1. Refer Ⅹ.Guide line for designing of primer. 4

5 IV. Storage: X One Step SYBR R RT-PCR Buffer IV should be protected from light. V. Features: (1) One Step RT-PCR made accurate and rapid analysis of RNA virus or small amount of RNA possible. (2) For PCR, enzymes for Hot Start TaKaRa Ex Taq TM HS, high efficiency hot start PCR enzyme, is used. Buffer system is optimized for Real-Time PCR, thus amplification is effective and high quality detection is possible. Moreover, One Step SYBR R RT-PCR Buffer Ⅳ is a 2x concentration of premix with SYBR R Green I, so the preparation of reactants is simple. VI. Note: Followings are protocol for this kit. Please read it carefully before you use the kit. (1) When mixing reagents for PCR, mix enough for 10 reactions for the master mix. Using master mixes allows accurate reagent dispensing, minimized reagent pipetting errors, and no repeat dispensing of the each reagent. This helps to minimize variation of the data from experiment to experiment. (2) PrimeScript TM 1step Enzyme Mix II should be mixed gently. Avoid generating bubbles! Gently spin down the solution prior to pipetting. Pipet the enzymes slowly as the enzyme contains 50% glycerol and is very viscous. (3) Keep the enzyme at - 20 until just before use and return into the freezer promptly after use. (4) Use new disposable pipette tips to avoid contamination between samples for transferring reagent. (5) Use the specific primer in this kit for reverse transcription. Random Primer or Oligo-dT Primer should not be used. 5

VII. Protocol: < Protocol using Thermal Cycler Dice Real Time System > 1. Prepare the following reagents on ice. < Per reaction > Reagents Volume Final Conc. 2X One Step SYBR R RT-PCR Buffer IV 12.

6 VII. Protocol: < Protocol using Thermal Cycler Dice Real Time System > 1. Prepare the following reagents on ice. < Per reaction > Reagents Volume Final Conc. 2X One Step SYBR R RT-PCR Buffer IV 12.5 μ l X PrimeScript TM 1 step Enzyme Mix II 1.0 μ l PCR Forward Primer (10 μ M) 1.0 μ l 0.4 μ M* 1 PCR Reverse Primer (10 μ M) 1.0 μ l 0.4 μ M* 1 total RNA 2.0 μ l * 2 RNase Free dh2o 7.5 μ l Total 25 μ l *1. The final concentration of primers can be 0.4 μ M in most reactions. If it does not work, determine the optimal concentrations within the range of μ M. *2. It is recommended to use 10 pg ng total RNA as templates. 2. Start the reaction Gently spin down the reaction tubes or plate with a centrifuge, then start the reaction after setting them onto Thermal Cycler Dice Real Time System * * Recommended to use standard protocol descried as follow for reaction. First, try this protocol; modify PCR reaction condition as needed. Use 3-step PCR if shuttle PCR is difficult to process, for example Primer with low Tm value. (Refer PCR reaction condition on page 10, for detail explanation.) Stage 1:Reverse Transcription Hold 42 5 min sec. Stage 2:PCR reaction Repeat:40 times 95 5 sec sec. Stage 3:Dissociation Note: This product combines the high performance of TaKaRa Ex Taq TM HS, which is an enzyme for hot start PCR utilizing Taq antibody. Heat inactivation step prior to PCR should be at 95 for 10 sec. No need to heat at 95 for (5-) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If longer heat treatment is done, the enzyme activity decreases and the amplification efficiency and the accuracy in quantification can also be affected. 6

7 3. After the reaction is completed, perform analysis. After the reaction is completed, verify the amplification curve and melting curve. Establish the standard curve when quantification is done. *Refer to the operation manual of Thermal Cycler Dice Real Time System and the following Experiment Examples for the analysis method with Thermal Cycler Dice Real Time System. < Protocol using ABI PRISM R 7000/7700 and Applied Biosystems 7300/7500 Real-Time PCR System > *Follow manuals of each instrument to operate them. 1. Prepare the following reagents on ice. < Per reaction > Reagents Volume Volume Final Conc. 2X One Step SYBR R RT-PCR Buffer IV 10 μ l 25 μ l 1X PrimeScript TM 1 step Enzyme Mix II 0.8 μ l 2 μ l PCR Forward Primer (10 μ M) 0.8 μ l 2 μ l 0.4 μ M* 1 PCR Reverse Primer (10 μ M) 0.8 μ l 2 μ l 0.4 μ M* 1 ROX Reference Dye or Dye II (50 X)* μ l 1 μ l total RNA 2 μ l 4 μ l* 2 * 2 RNase Free dh2o 5.2 μ l 14 μ l Total 20 μ l* 4 50 μ l* 4 *1. The final concentration of primers can be 0.4 μ M in most reactions. If it does not work, determine the optimal concentrations within the range of μ M. *2. It is recommended to use 20 pg ng total RNA as templates in 50 μ l reaction. *3. The ROX TM Reference Dye/Dye II is supplied for performing normalization of fluorescent signal intensities among wells when used with real time PCR instruments that have option. For ABI PRISM R 7000/7700 and Applied Biosystems 7300 Real-Time PCR System, the use of ROX TM Reference Dye (50X) is recommended. For Applied Biosystems 7500 Real-Time PCR System, the use of ROX TM Reference Dye II is recommended. *4. The reaction of 50 μ l is for 96-well plate, single tube and 8-strip tube. The reaction of 20 μ l is for 384-well plate. 7

2. Start reaction Recommended to use standard protocol descried as follow for reaction. First, try this protocol; modify PCR reaction condition as needed.

8 2. Start reaction Recommended to use standard protocol descried as follow for reaction. First, try this protocol; modify PCR reaction condition as needed. Use 3-step PCR if shuttle PCR is difficult, for example Primer with low Tm value. Stage 1,2:Reverse transcription Reps: min sec. Stage 3:PCR reaction Reps: sec ~34 sec. * Stage4:Dissociation Protocol *Set up at 30 seconds for 7700, 31 seconds for 7000 and 7300, and 34 seconds for (Refer PCR reaction condition on page 10, for detail explanation.) Note: This product combines the high performance of TaKaRa Ex Taq TM HS, which is an enzyme for hot start PCR utilizing Taq antibody. Heat inactivation step prior to PCR should be at 95 for 10 sec. No need to heat at 95 for (5-) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If longer heat treatment is done, the enzyme activity decreases and the amplification efficiency and the accuracy in quantification can also be affected. 3. After the reaction is completed, verify the amplification curve and dissociation curve. Establish the standard curve when quantification is done. *Refer to the operation manual of an used real time PCR instrument. 8

< Protocol using LightCycler R > * Follow manual of LightCycler R (by Roche Diagnostics) to operate it. 1. Prepare the following reagents on ice. < Per reaction > Reagents Volume Final Conc.

9 < Protocol using LightCycler R > * Follow manual of LightCycler R (by Roche Diagnostics) to operate it. 1. Prepare the following reagents on ice. < Per reaction > Reagents Volume Final Conc. 2X One Step SYBR R RT-PCR Buffer IV 10 μ l X PrimeScript TM 1 step Enzyme Mix II 0.8 μ l PCR Forward Primer (10 μ M) 0.8 μ l 0.4 μ M* 1 PCR Reverse Primer (10 μ M) 0.8 μ l 0.4 μ M* 1 total RNA 2 μ l * 2 RNase Free dh2o 5.6 μ l Total 20 μ l *1. The final concentration of primers can be 0.4 μ M in most reactions. If it does not work, determine the optimal concentrations within the range of μ M. *2. It is recommended to use 10 pg ng total RNA as templates. 2. Start reaction Gently spin down PCR capillaries, then start the reaction after setting them onto LightCycler R.* *Recommended to use standard protocol descried as follow for reaction. First, try this protocol; modify PCR reaction condition as needed. Use 3-step PCR if shuttle PCR is difficult, for example Primer with low Tm value. (Refer PCR reaction condition on page 11, for detail explanation.) Stage 1:Reverse transcription 42 5 min. 20 /sec sec. 20 /sec. 1 Cycle Stage 2:PCR reaction 95 5 sec. 20 /sec sec. 20 /sec. 40 Cycle Stage 3:melting curve analysis 95 0 sec. 20 /sec sec. 20 /sec sec. 0.1 /sec. 9

10 Note: This product combines the high performance of TaKaRa Ex Taq TM HS, which is an enzyme for hot start PCR utilizing Taq antibody. Heat inactivation step prior to PCR should be at 95 for 10 sec. No need to heat at 95 for (5-) 15 min. as the initial denaturation, required for chemically modified Taq polymerase. If longer heat treatment is done, the enzyme activity decreases and the amplification efficiency and the accuracy in quantification can also be affected. 3. Analyze after completion of reaction. After the reaction is completed, verify amplification curve and melting curve. Establish the standard curve when quantitative analysis is necessary. * *Refer to the operation manual of LightCycler R. PCR Reaction Condition Shuttle PCR Step Temp. Time Detection Remark Denature 95 3~5 sec. Off Since the target size amplified for real time PCR is generally shorter than 300 bp, the denaturation at 95 for 3-5 seconds is a sufficient condition. Annealing/ 60~66 20~30 sec. On Please try at 60 for 20 seconds Extension (30~34 sec.)* 1 at first. The temperature should be optimized within the range of if optimization is required. When reaction does not proceed efficiently, extened the time or change the reaction into 3 step PCR. 3 step PCR Step Temp. Time Detection Remark Denature 95 3~5 sec. Off Since the target size amplified for real time PCR is generally shorter than 300 bp, the denaturation at 95 for 3-5 seconds is a sufficient condition. Annealing 55~60 10~20 sec. Off Please try at 55 for 10 seconds at first. When non-specific amplified product generate or when the amplification efficiency is low, it can be solve with the optimization of annealing temperature. Longer annealing time may sometimes improve the amplification efficiency. Extension 72 6~15 sec. On When the amplified size is less (30~34 sec.)* 1 than 300 bp, the time should be determined within the range of 6-15 seconds. Longer extension time can cause nonspecific amplification. Cycle: 30~45cycles 10 *1: Detection step must be set at more than 30 seconds for instruments of Applied Biosystems. For 7700 should be set at 30 seconds, 7000 and 7300 should be set at 31 seconds, and 7500 should be set at 34 seconds.

VIII. Experiment Example: Detection of Human HPRT1 (hypoxanthine phosphoribosyl transferase1) (Thermal

4 pg~100 ng that was prepared from Human Liver and sterilized water (negative control) as an template,

Result Amplification Curve Melting Curve After reaction, Ct value from 2nd derivative of amplification

Discussion Target DNA were detected using total RNA 6.4 pg - 100 ng as templates.

11 VIII. Experiment Example: Detection of Human HPRT1 (hypoxanthine phosphoribosyl transferase1) (Thermal Cycler Dice TM Real Time System is used here) 1. Procedure Using total RNA 6.4 pg~100 ng that was prepared from Human Liver and sterilized water (negative control) as an template, Real-Time One Step RT- PCR was performed. 2. Result Amplification Curve Melting Curve After reaction, Ct value from 2nd derivative of amplification curve was obtained, and the Standard curve was created. 2nd derivative Standard Curve 3. Discussion Target DNA were detected using total RNA 6.4 pg ng as templates. The melting curve shows that the same amplification products were obtained even when different amount of template were used. The linearity of standard curve was obtained within the wide range concentration of template. 11

12 IX. Appendix: A. Preparation of RNA sample This kit is designed to perform the reverse transcription of RNA to cdna and subsequent amplification. It is important to use high purity of RNA sample for better yields of cdna synthesis. So, it is essential to inhibit cellular RNase activity and also to prevent contamination with RNase derived from equipment and solutions used. Extra precaution should be taken during the sample preparation, including use of clean disposable gloves, dedication of a table exclusively for RNA preparation, and avoiding unnecessary speaking during assembly, to prevent the RNase contamination from operates sweat or saliva. [ Equipment ] Disposable plastic equipment shall be used. Glass tools should be treated with either of the following protocol prior to use. (1) Hot-air sterilization (180, 60min). (2) Treatment with 0.1% DEPC at 37 for 12 hours, followed by autoclaving at 120 for 30 min. to remove DEPC. * It is recommended that all the equipment be used as the exclusive use 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, 60min) or DEPC treatment], and distilled water must be treated with 0.1%DEPC and autoclaved. All reagents and distilled water should be used exclusively for RNA experiments. [ Preparation of RNA sample ] It is recommended to use highly purified RNA obtained by GTC (Guanidine thiocyanate) method, etc. 12

13 X. Guidline for designing of primer: It is essential to design primers which allow good reactivity for a successful real time PCR reaction. Please follow the guideline stated as below to design primers which offer high amplification efficiency and minimizes non-specific reaction. Amplification product Amplified size bp is most recommended. (Possible to amplify a target up of 300 bp.) Primer Length 7-25 mer GC content 40-60% (45-55% is recommended.) Tm Tm values of Forward primer and Reverse primer must not largely differ. Tm value is calculated with the software for calculation of Tm value. e.g. OLIGO *1 : Primer 3 *2 : Sequence The sequence should not be partially rich in any base in the whole region. Avoid including parts which has high GC or AT content (especially 3' -end). Not include polypyrimidine (serial T/C sequence). Not include polypurine (serial A/G sequence). Sequence of 3' end The termini part of 3' should not have high content of GC or AT. It is recommended to be G or C at 3' end. It is not recommended to be T at 3' end. Complementarity Complementary sequence of more than 3 bases should not exist within a primer or even between primer pairs. Primer pair should not have a complementary sequence of more than 2 bases at the 3' end each. Specificity Specificity of primers should be confirmed through BLAST search. *3 *1: OLIGO TM Primer Analysis Software (Moleculor Biology Insights, Inc.) *2: Primer3 ( *3: 13

14 XI. Related Products : One Step PrimeScript TM RT-PCR Kit (TaKaRa Cat.#RR064A) SYBR Premix Ex Taq TM (TaKaRa Cat.#RR041A) Premix Ex Taq TM (TaKaRa Cat.#RR039A) FastPure TM RNA Kit (TaKaRa Cat.#9190) NOTE: This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from If you require licenses for other use, please call at or contact from our website at com. NOTICE TO PURCHASER: LIMITED LICENSE [P5] PCR Notice Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [M57] LA Technology This product is covered by the claims 6-16 of U.S. Patent No. 5,436,149 and its foreign counterpart patent claims. [L1] One Step RNA PCR / One Step RT-PCR Use of this product is licensed from biomerieux, is covered by US Patent 5,817,465 and equivalents, and is for Research Use Only. [L11] SYBR Green I This product is covered by the claims of U.S. Patent No. 5,436,134 and 5,658,751 and their foreign counterpart patent claims. Takara PCR products containing SYBRR Green I are sold under license from Molecular Probes Inc. only for the usage in Real-time PCR for internal research purpose. These products are not to be used for the purpose such as; providing medical, diagnostic, or any other testing, analysis or screening services or providing clinical information or clinical analysis in return for compensations. [L15] Hot Start PCR Licensed under U.S. Patent 5,338,671 and 5,587,287 and corresponding patents in other countries. Smart Cycler R is a trademark of Cepheid. 14 Phone: Fax:

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

Cat. # RR086A For Research Use One Step SYBR PrimeScript RT-PCR Kit II Product Manual Table of Contents I. Description...3 II. III. IV. Principle...3 Components...5 Storage...6 V. Features...6 VI. VII.