BcaBEST TM RNA PCR Kit Ver.1.1

Transcription

1 Table of Contents I. Description... 2 II. Principle... 4 III. Features... 5 IV. Note... 6 V. Protocol... 7 VI. Application examples... 9

2 Ⅰ escri tion. escri tion Description: PCR(Polymerase Chain Reaction) process is a simple and powerful method which allows in vitro amplification of DNA fragments through a succession of three incubation steps at different temperatures. In principle, PCR is a method to amplify DNA segments, and not directly amplify RNA. However, synthesis of cdna from RNA using reverse transcriptase enables to apply PCR process to the RNA analysis. Many reports of various fields have been made by applying this method, such as of structual analysis of RNA, efficient cdna cloning, analysis of gene expression at the RNA level, etc. BcaBEST TM RNA PCR Kit is designed to perform the reverse transcription of RNA to cdna using BcaBEST TM Polymerase and subsequent amplification using Bca-Optimized Taq all in a single tube. BcaBEST TM Polymerase has both DNA polymerase activity, and reverse transcriptase activity, isolated from Bacillus caldotenax. Bca-Optimized Taq is especially constructed by Takara utilizing the advantage of LA Technology.* Including these two specially designed enzymes, this kit is supplied with all the necessary reagents for reverse transcription and PCR. BcaBEST TM RNA PCR Kit is very useful for synthesizing cdna from RNA forming highly secondary structure because the optimum reaction temperature of RT- PCR using BcaBEST TM Polymerase is 65, higher than that of the conventional reverse transcriptases. This version 1.1 is an improved kit of the old one to work more effectively for a highly-structured template. (Refer to Application example, page 9-11 ). *U.S. Patent 5,436,149 for LA Technology is owned by Kit components (100 reactions*): One reaction is 10 µl RT followed by 50 µl PCR. 1. BcaBEST TM Polymerase (22 units/µl) 50 µl (originated from Bacillus caldotenax) 2. RNase Inhibitor (40 units/µl) 25 µl 3. Random 9 mers** (50 µm) 50 µl 4. Oligo dt Primer** (50 µm) 50 µl 5. RNase Free distilled H 2 O ml 6. Bca-Optimized Taq (5 units/µl) 25 µl 7. 2x Bca 1st Buffer 2 x 1.25 ml 8. 5x Bca 2nd Buffer 800 µl 9. dntp Mixture (ea. 10 mm) 50 µl 10. MgSO 4 (25 mm) 500 µl 11. Control F-1 primer*** (20 µm) 25 µl (upstream sense primer for positive control RNA) 12. Control R-1 primer*** (20 µm) 25 µl (downstream antisense primer for positive control RNA) 13. Positive control RNA*** (2 x 10 5 copies/µl) 25 µl * This kit is designed for 100 reactions. (the total reaction volume; RT reaction 10 µl, PCR 50 µl) In the previous protocol of this kit, it was designed for 50 reactions. (the total reaction volume; RT reaction 20 µl, PCR 100 µl) The number of reaction is changed as mentioned above, but the volume of all component in this kit is not changed. **Primers Sequence Random 9 mers: -NNNNNNNNN- Oligo dt Primer: The original oligo dt primer designed by Takara.This primer does not include the region complementary to M13 Primer M4, and is different from Oligo dt Adaptor primer which is supplied in TaKaRa RNA PCR Kit Ver.3.0 (#RR019) and TaKaRa RNA LA PCR Kit Ver.1.1(#RR012). Control F-1 primer: -CTGCTCGCTTCGCTACTTGGA- Control R-1 primer: -CGGCACCTGTCCTACGAGTTG- 2

3 ***Positive control RNA Supplied control RNA is in vitro transcribed RNA using SP6 RNA polymerase from plasmid psptet3 inserted with DNA fragment (approximately 1.4 kb) having tetracycline resistant gene, originated from pbr322, in the downstream of SP6 promoter. This control RNA is a poly(a) + RNA containing 30 bases of poly(a) at the tail. When full-length double-stranded cdna is synthesized from this control RNA, tetracycline resistant plasmid is obtained by inserting this cdna. S P6 Promoter Tetracycline R esista nce G ene Hind III III BamH B I S Sal I EcoR E V SSph I I F -1 Primer R -1 P rimer AAAA bp Fig. 1 Amplified DNA fragment using control RNA and control primers Reagents not supplied in the kit: 1. Agarose gel ex. NuSieve R 3:1 Agarose (Lonza Biosciences) Equipment required: 1. Authorized thermal cyclers (with slope function) ex. TaKaRa Thermal Cycler Dice TM (Takara Cat. #TP600) 2. Agarose gel electrophoresis apparatus 3. Microcentrifuge 4. Micropipets and pipette tips (autoclaved) Storage: -20 References: 1) Kawasaki, E. S. and Wang, A. M. (1989) PCR Technology (Erlich, H. A. ed.), Stockton Press, ) Lynas, C., Cook, S. D., Laycock, K. A., Bradfield, J. W. B., and Maitland, N. J. (1989) J. Pathology, 157, ) Frohman, M. A., Dush, M. K., Martin, G. R. (1988) Proc. Natl. Acad. Sci. USA, 85,

4 Ⅱ Princi le Principle: Fig.2 Schematic diagram of RNA PCR mmrna R N A SSynthesis of of first first strand strand cdna cd N A wwith BBcaBEST E SPolymerase T P erase cdcdna N A PPCR C R components ponents added; added; SSecond strand strand cdna cd Nsynthesized A synthesized with w ith B ca O ptim ized Taq Bca-Optimized Taq Amplify cdna A m plify cd N A Flow chart of RNA PCR mrna Prepare reaction mixture by combining BcaBEST TM Polymerase and other reagents necessary for reverse transcription. Reaction condition: 65 min min. Gradually raise the temperature to 65 over min.* min. 1 cycle 98 5 min. 5 5 min. *Some templates do not require the gradual raise of temperature. Add Bca-Optimized Taq and other reagents necessary for PCR into the same tube. Condition of thermal cycling: sec sec cycles 72-5 min. PCR products Perform agarose gel electrophoresis to verify the amplified products. 4

5 BcaBEST TM RNA PCR Kit allows reverse transcription from RNA to cdna using Bca- BEST TM Polymerase and subsequent amplification in the same tube utilizing Bca- Optimized Taq. Random 9 mers, Oligo dt Primer, or a specific downstream primer which act as an anti-sense primer in PCR process can be used for cdna synthesis. Ⅲ Features Features: Template RNA RNA segment to be transcribed and amplified Reverse Transcriptase DNA Polymerase RNase Inhibitor Primer for 1st strand cdna synthesis Protocol For all samples (Especially effective for mrna with high GC content or with a complex structure.) at least 5 kbp BcaBEST TM Polymerase (Optimum reaction temperature: 65 ) Bca-Optimized Taq Supplied in the kit Random 9 mers, or Oligo dt Primer or Specific downstream PCR primer Single tube reaction (RTase is heat inactivated prior to PCR) Preparation of RNA sample : BcaBEST TM RNA PCR Kit is designed to perform the reverse transcription of RNA to cdna and subsequent amplification. The purity of RNA sample will affect the yield of cdna synthesis. So it is essential to inhibit the activity of RNase in the cells and also to prevent the contamination of RNase derived from equipments and solutions used. Extra precautions should be taken during the sample preparation; put on clean disposable gloves, dedicate a table to exclusive use for RNA preparation, and avoid unnecessary talks during the operations to prevent the contamination of RNase from operators' sweat or saliva. A. Equipment Disposable plastic equipments shall be used. In case using glass tools, treat the glass tools with DEPC(diethylpyrocarbonate) prior to use. (1) Treat glass tools with 0.1% DEPC solution at 37, 12 hours. (2) Autoclave at 120, 30min. to remove DEPC. It is recommended to prepare all the equipments as the exclusive use for RNA preparation. B. Reagent Reagents for RNA preparation, including distilled water, shall be prepared with heat sterilized glass tools (180, 60 min.), if possible those treated with 0.1% DEPC solution and autoclaved. Reagents and distilled water should be exclusively used for RNA preparation. C. Preparation method of RNA sample Simple purification methods can yield enough amount of RNA for reverse transcription and subsequent PCR. However, it is recommended to use highly purified RNA obtained by GTC(Guanidine thiocyanate) method, etc. D. RNA Sample Amount Approximately 500 ng of total RNA is appropriate per one reaction. 5

6 Ⅳ N Note: 1) For both reverse transcription and PCR amplification, master mix of reagents(containing RNase-free sterilized distilled water, buffers, dntp mixtures, MgSO 4 solution, etc) for all samples can be prepared first, then aliquoted to individual tubes. Using such mixtures will allow accurate reagents dispense: minimize reagents pipetting losses, and avoid repeat dispensing of the each reagent. This helps to minimize variation of the data among the experiments. 2) Enzymes such as RTase (BcaBEST TM Polymerase), Bca-Optimized Taq, and RNase Inhibitor shall be mixed gently by pipetting. Avoid generating bubbles. Gently spin down the solution prior to mixing. Pipette enzymes carefully and slowly as the viscosity of the 50% glycerol in the enzyme solution can lead to pipetting errors. 3) Keep enzymes 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. 5) PCR condition Optimum PCR condition varies depending on an used thermal cycler. It is recommended to perform a control experiment to determine the condition prior to using a sample. 6) Primer Selection Depend on many factors, the primer for reverse transcription should be selected from either of Random 9 mers, Oligo dt primer or specific downstream PCR primer. For short mrnas with no hairpin structure, any one of the above three primers can be used. [ General guideline of the primer selection ] Random 9 mers: Use for long transcription of long RNAs or of RNA with hairpin structure. Also can be used to reverse transcribe all RNA (rrna, mrna and trna). Any pairs of PCR primers work equally well in PCR of cdna synthesized with Random 9 mers. Specific downstream primer (anti-sense primer in PCR) Use for the target RNA which sequence is already determined. Oligo dt Primer Use only for mrnas with poly(a) tails (Note: Prokaryotic RNA, eukaryotic rrna and trna, and some eukaryotic mrna do not have poly(a) tails) This primer was designed originally by Takara for efficient cdna synthesis. 6

7 Ⅴ Protocol Protocol: Typical RT-PCR example A. Reverse Transcription 1. Prepare the reaction mixture in a tube by combining the reagents in the proportions shown. The primer for a cdna synthesis should be chosen from either of Random 9 mers, Oligo dt primer or specific downstream primer. For the control experiment, use R-1 primer. (See "Note (6) Primer Selection" for selection of primer to use.) Reagents Volume Final concentration 2x Bca 1st Buffer 5 µl 1X 25 mm MgSO 4 2 µl 5 mm dntp Mixture (10 mm) 0.5 µl 0.5 mm RNase Inhibitor (40 units/µl) 0.25 µl 1 unit/µl BcaBEST TM Polymerase (22 units/µl) 0.5 µl 1.1 units/µl Oligo dt-primer or Random 9 mers 0.5 µl or Specific downstream PCR primer Positive control RNA [ 1 x 10 5 copies] or 0.5 µl Experimental Sample* or [ 500 ng total RNA] RNase Free distilled H 2 O 0.75 µl Total volume 0 µl per sample *When using low expression sample RNA, the volume can be increased up to µl. 2. If necessary overlay mineral oil to avoid the evaporation of the reaction mixture. 3. Place all tubes in a Thermal Cycler and perform the reaction with the program as below* min 30 1 min Gradually raise to min min 98 5 min 5 5 min *In case of using Positive control RNA, the gradual temperature raise is performed over 15 min, and incubation time at 65 is 15 min. 7

8 B. PCR Two kinds of reaction mixture are available for BcaBEST TM RNA PCR Kit Ver.1.1; A and B methods. A method is employed in general reactions. If an amplification is not successful with A method, B method is recommended. 1. Prepare reaction mixture by combining the following reagents*. (This is "A method") When the reaction mixture is added into the RT reactant for the subsequent PCR, the mixture should not contact with mineral oil. It is recommended to pour the mixture with a pipette into the underlayer of mineral oil. Reagents Volume Final conc. in PCR (per 50 µl mixture) MgSO 4 (25 mm) 3 µl 2.5 mm 5x Bca 2nd Buffer 8 µl 1x Bca-Optimized Taq 0.25 µl 1.25 units/50 µl Upstream sense PCR Primer**(20 µm) 0.5 µl 0.2 µm (F-1 Primer for Control RNA) Downstream antisense PCR Primer**(20 µm) 0.5 µl 0.2 µm (R-1 Primer for Control RNA) Sterilized distilled water µl Total volume 40 µl per sample *B method In case that an amplified fragment can not be obtained with the above A method, it is recommended to perform PCR again by preparing a reaction mixture with the following composition. This reaction mixture is especially effective for amplification of a template with a complex structure. Reagents Volume Final conc. in PCR (per 50 µl mixture) MgSO 4 (25 mm) 3 µl 2.5 mm 2x Bca 1st Buffer 20 µl 1x Bca-Optimized Taq 0.25 µl 1.25 units/50 µl Upstream sense PCR Primer**(20 µm) 0.5 µl 0.2 µm (F-1 Primer for Control RNA) Downstream antisense PCR Primer**(20 µm) 0.5 µl 0.2 µm (R-1 Primer for Control RNA) Sterilized distilled water 5.75 µl Total volume 40 µl per sample 2. Add 40 µl of the mixture into a tube containing the cdna obtained at A. 3. Spin for approximately 10 seconds with a microcentrifuge. 4. Place the tubes in a Thermal Cycler and perform ampification under the optimal condition.*** General PCR condition 94, 30 sec , 30 sec cycles 72, 1-10 min min. PCR condition for Positive control RNA 94, 30 sec. 60, 30 sec. 28 cycles 72, 1 min. 8

9 **Primers and amplified DNA when using the supplied Control RNA Primer for PCR Primers Amplified fragment reverse transcription Oligo dt Primer F-1,R bp Random 9 mers F-1,R bp R-1 Primer F-1,R bp *** Conditions for PCR 1) Annealing Temperature: 60 is optimal for amplification of control RNA. It may be necessary to lower or raise the annealing temperature for RNA samples. (The optimal temperature need to be determined empirically by testing temperatures within the range of 37 to 65 ). 2) Extension time: The length of the target sequence will affect the required extension time. Typically, Bca-Optimize Taq has an extension rate of 1~2 min./1000 bases at 72. 3) Number of cycles: When small volume of cdna is used, the repetition of 30~50 cycles are required for PCR amplification. 5. After the amplification is completed, apply 5~10 µl of the reactant for agarose gel electrophoresis to verify the amplified DNA fragments.**** ****The PCR amplified product samples can be stored frozen until subsequent analysis. Ⅵ A lication Application exam les examples: The RT-PCR performances were compared between BcaBEST TM RNA PCR Kit Ver.1.1 (#RR023) and other Takara' s RNA PCR kits (AMV) using two kinds of template with highly secondary structure. Example 1: Amplification of TRADD gene (Comparison between BcaBEST TM RNA PCR Kit Ver.1.1(#RR023) vs TaKaRa RNA PCR Kit Ver.2.1 (AMV) (#R019)) Target: human TRADD gene 494 bp ( Total GC content: 68.2%) Sample: Total RNA derived from HeLa cell Primer for RT reaction: Oligo-dT primer Protocol: The RT-PCR reactions were performed under the optimal conditions by preparing optimum reaction mixture, respectively, according to each kit. Reverse transcription condition BcaBEST TM RNA PCR Kit Ver.1.1(#RR023) 65, 1 min. 30, 5 min. gradually raise up to 65 over 15 min. 65, 30 min. 98, 5 min. 5, 5 min. RNA PCR Kit Ver.2.1 (#R019) 30, 5 min. 50, 30 min. 98, 5 min. 5, 5 min. PCR condition (for all kits) 94, 1 min. 94, 30 sec. 60, 30 sec. 30 cycles 72, 1 min. 72, 5 min. 9

10 Result: BcaBEST TM RNA PCR Kit Ver.1.1 achieved amplification of TRADD gene, though RNA PCR Kit Ver.2.1 could not. M 1 2 M Lane: M: 100 bp DNA ladder : RNA PCR Kit Ver.2.1 2: BcaBEST TM RNA PCR Kit Ver.1.1 M: 100 bp DNA ladder 494 bp 3% Agarose gel Applied volume: 4 µl / lane Example 2: Amplification of IGFR II gene (template with a more complex structure) (Comparison among BcaBEST TM RNA PCR Kit Ver.1.1 (#RR023), RNA PCR Kit Ver.2.1(AMV) (#R019) and RNA LA PCR Kit Ver.1.1(AMV) (#RR012)) Target: IGFR II 560 bp (Total GC content: 63.5%) Sample: Total RNA derived from human skeletal muscles Protocol The RT-PCR reactions were performed under the optimal conditions by preparing optimal reaction mixture, respectively, according to each kit. For BcaBEST TM RNA PCR Kit Ver.1.1, two kinds of PCR reaction mixture. (A method and B method) were prepared. RT condition BcaBEST TM RNA PCR Kit Ver.1.1(#RR023) 65, 1 min. 30, 5 min. gradually raise up to 65 over 15 min. 65, 15 min. 98, 5 min. 5, 5 min. RNA LA PCR Kit Ver.1.1(#RR012) and RNA PCR Kit Ver.2.1 (#R019) 30, 5 min. 50, 30 min. 98, 5 min. 5, 5 min. PCR condition (for all kits) 94, 1 min. 94, 30 sec. 60, 30 sec. 40 cycles 72, 1 min. 72, 5 min 10

11 Result: Only B method of BcaBEST TM RNA PCR Kit Ver.1.1 achieved amplification, though A method and the other 2 kits could not yield PCR products of interest (516 bp). M M 516 bp Lane: M: 100 bp DNA ladder 1: A method of BcaBEST TM RNA PCR Kit Ver.1.1 2: B method of BcaBEST TM RNA PCR Kit Ver.1.1 3: RNA PCR Kit Ver.2.1 4: RNA LA PCR Kit Ver.1.1 3% Agarose gel Conclusion: - The example 1 shows that BcaBEST TM RNA PCR Kit Ver.1.1 is more effective than RNA PCR Kit Ver.2.1(AMV) for amplification of templates which have high GC content. - The example 2 shows that B method of BcaBEST TM RNA PCR Kit Ver.1.1 enables RT-PCR when using a further highly-structured template, by utilizing 2x Bca 1st Buffer in PCR reaction. 11

12 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 NOTICE TO PURCHASER: LIMITED LICENSE [M21]BcaBEST DNA Polymerase This product is covered by the claims of U.S. Patent Nos. 5,436,326 and 5,753,482 and their foreign counterpart patent claims. [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. 12 Phone: Fax: