Table of Contents. I. Description II. Kit Components III. Storage IV. 1st Strand cdna Synthesis Reaction... 3

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1 Table of Contents I. Description... 2 II. Kit Components... 2 III. Storage... 2 IV. 1st Strand cdna Synthesis Reaction... 3 V. RT-PCR, Real-time RT-PCR... 4 VI. Application... 5 VII. Preparation of RNA Sample... 7 VIII. Related Products... 8 URL: TAKARA BIO INC. 1

2 I. Description: BluePrint T 1st Strand contains all the reagents necessary to synthesize 1st strand cdna from total or poly(a) + RNA using BluePrint T RTase. BluePrint T is a newly developed LV-based RTase. This enzyme shows excellent elongation ability and is capable of synthesis of cdna up to 12 kb in length. Also, it can synthesize cdna efficiently at standard reverse transcription temperatures (42 C), even when RNA templates contain GC- rich regions or difficult-to-synthesize structures. This avoids high temperature reactions which may cause RNA decomposition. 1st strand cdnas synthesized with this kit can be used for variety of applications including 2nd strand synthesis, hybridization, PCR amplification, and real-time PCR. II. Kit Components: (for 10 reactions) BluePrint T 1st Strand RTase 5X BluePrint T 1st Strand Buffer Recombinant RNase Inhibitor (40 U/ μ l) dntp ixture (10 m each) Oligo dt Primer (50 μ ) Random 6 mers (50 μ ) RNase Free H 2O 10 μ l 40 μ l 5 μ l 10 μ l 10 μ l 20 μ l 1 ml Sequence of each primer Primer Sequence Random 6 mers pd(n) 6 Oligo dt Primer TaKaRa Bio originally designed dt sequence region * 1 *1. This sequence is different from the Oligo dt Adaptor Primer supplied with the TaKaRa RNA PCR Kit (AV) Ver It does not contain the 13 Primer 4 complimentary region. Reagents and Instrument not supplied in this kit 1. DNA Amplification System (authorized instruments) TaKaRa PCR Thermal Cycler Dice T, etc Water bath 2. Agarose L03 (TaKaRa Cat.# 5003) 3. Electrophoresis Apparatus 4. icrocentrifuge 5. icropipetts and pipette tips (autoclaved) III. Storage: 20 2 TAKARA BIO INC. URL:

3 IV. 1st-Strand cdna Synthesis Reaction: [Standard Protocol] 1. Prepare the following mixture in a microtube. Reagent Volume Oligo dt Primer (50 μ ) 1 μ l or Random 6 mers (50 μ ) 1 μ l (0.4-2 μ l)* 1 dntp ixture (10 m each) 1 μ l Template RNA total RNA : less than 5 μ g* 2 poly(a) + RNA : less than 1 μ g RNase free dh 2O up to 10 μ l 2. Incubate for 5 minutes at 65 C; cool immediately on ice. 3. Prepare the reaction mixture by combining the following reagents to a total volume of 20 μ l. Reagent Template RNA Primer ixture 5X BluePrint T 1st Strand Buffer Recombinant RNase Inhibitor (40 U/ μ l) BluePrint T RTase RNase free dh 2O Volume 10 μ l 4 μ l 0.5 μ l 1.0 μ l up to 20 μ l 4. ix gently 5. Incubate the reaction mixture immediately under the following conditions 30 C 10 min (Only when Random 6 mers are used.) 42 C (50 C)* min. 6. Inactivate the enzymes by incubation at 95 C for 5 minutes* 4, followed by cooling on ice. *1. For best results, Takara Bio recommends use of 0.4 μ l (20 pmol) Random 6mers for synthesis of cdna over 2 kb long using random primers. Use 2 μ l (100 pmol) for synthesis of real-time PCR templates using random primers. Also, if a Gene Specific Primer is used, the final primer concentration should be 0.1 μ. *2. For 1st strand cdna synthesis of a real-time RT-PCR template, the total RNA amount should be less than 1 μ g. *3. General reactions should be performed at 42 C because of BluePrint T RTase s strong extension through higher-order structures. If using a specific downstream PCR primer as the reverse transcription primer, there may be some non-specific amplification due to mispriming. In this case, raising the reaction temperature to 50 C may improve the result. *4. For amplification of longer targets, enzyme inactivation at 70 C for 15 min. is recommended to minimize damage to the 1st strand cdna (i.e. nicking). URL: TAKARA BIO INC. 3

4 V. RT-PCR, Real-time RT-PCR The 1st Strand cdna synthesis reaction mix can be used directly as the PCR or real-time PCR template without purification. However, the volume of the 1st strand reaction must be less than 1/10th of the total PCR reaction volume. Also, there are PCR enzymes for which the rate of amplification may be affected by the amount of starting template. Thus, please refer to PCR enzyme instruction manual to assess the appropriate amount of template to use. In case of non-specific amplification or no product produced after amplification, results can be improved by treating the cdna synthesis reaction with RNase H. Recommended PCR enzymes For excellent efficient PCR: TaKaRa Ex Taq T, TaKaRa Ex Taq T HS For long PCR: TaKaRa LA Taq T, TaKaRa LA Taq T HS For Accurate PCR: PrimeSTAR T HS DNA Polymerase Recommended real-time PCR reagents SYBR R Green I detection: SYBR R Premix Ex Taq T (Perfect Real Time), Taqan R Probe detection: Premix Ex Taq T (Perfect Real Time) 4 TAKARA BIO INC. URL:

5 VI. Application (1) Using varying amounts of HL60 total RNA as a template, a human 28S ribosomal RNA (418 bp, GC 70%) target was reverse transcribed using a gene specific primer (0.1 µ) and the Blue- Print 1st Strand at 42 C (20 µl reaction). 5 µl of the RT reaction mix was then amplified by PCR, with or without prior RNase H treatment. The results demonstrate that highly sensitive cdna synthesis and subsequent amplification of this high GC target were achieved (down to 1 pg Total RNA) using this kit. RNase H treatment prior to PCR is not required for high-efficiency amplification. RNase H RNase H : 100 bp DNA Ladder 1: 1 pg HL60 Total RNA 2: 10 pg HL60 Total RNA 3: 100 pg HL60 Total RNA 4: 1 ng HL60 Total RNA 5: 10 ng HL60 Total RNA 6: 100 ng HL60 Total RNA 7: 1 µg HL60 Total RNA : 100 bp DNA Ladder Efficient cdna amplification was confirmed with and without RNase H treatment. URL: TAKARA BIO INC. 5

6 (2) Using varying amounts of HL60 total RNA as a template, an ApoE 520 bp target (GC 74.8%) was reverse transcribed using an Oligo dt primer (2.5 µ) and the BluepPrint 1st Strand at 42 C (20 µl reaction). 5 μl of the RT reaction mix was then amplified by PCR, with or without prior RNase H treatment. The results demonstrate that highly sensitive cdna synthesis and subsequent amplification of this high GC target were achieved (to 10 ng Total RNA) using this kit. RNase H treatment prior to PCR is not required for high-efficiency amplification. RNase H - RNase H : 100 bp DNA Ladder 1: 100 pg HL60 Total RNA 2: 1 ng HL60 Total RNA 3: 10 ng HL60 Total RNA 4: 100 ng HL60 Total RNA 5: 1 µg HL60 Total RNA :100 bp DNA Ladder Efficient cdna amplification was confirmed with and without RNase H treatment. 6 TAKARA BIO INC. URL:

7 VII. Preparation of RNA sample It is important to use highly pure RNA samples for better cdna yield. It is essential to inhibit cellular RNase activity and also to prevent contamination with RNase derived from equipment and/ or solutions. Extra precautions should be taken during sample preparation, including use of clean disposable gloves, dedication of a table exclusively for RNA preparation, and avoiding unnecessary speaking during assembly, to prevent RNase contamination from user sweat or saliva. [Equipment] Disposable plastic equipment should be used. Glass tools should be treated with the following protocol prior to use. (1) Hot-air sterilization (180 C, 60min) (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 autoclaved. All reagents and distilled water used should be exclusively for RNA experiments. [Preparation of RNA sample] Use of highly purified RNA purified by the GTC (Guanidine thiocyanate) method, etc is recommended. It is possible to obtain highly purified total RNA by using FastPure RNA Kit (TaKaRa Cat.# 9190) for cultured cells or tissue samples. RNA samples should be suspended in either autoclaved distilled water or TE Buffer solution prior to reverse transcription. URL: TAKARA BIO INC. 7

8 VIII. Related Products: RT-PCR kit For highly efficient and sensitive 2 step RT-PCR BluePrint RT-PCR Kit (TaKaRa Cat.# RR714A) For highly sensitive 1 step RT-PCR BluePrint One Step RT-PCR Kit (TaKaRa Cat.# RR755A) For longer and more accurate RT-PCR products: RNA LA PCR Kit, Version 1.1 (TaKaRa Cat.# RR012A) For real time RT-PCR: One Step Ex Taq qrt-pcr Kit (TaKaRa Cat.# RR067A/B) PrimeScript RT Reagent Kit (Perfect Real Time) (TaKaRa Cat.# RR037A/B) One Step RT with SYBR for Real Time RT-PCR: One Step SYBR Ex Taq qrt-pcr Kit (TaKaRa Cat.# RR019A/B) PCR Enzyme For efficient PCR: TaKaRa Ex Taq (TaKaRa Cat.# RR001A/B), TaKaRa Ex Taq HS (TaKaRa Cat.# RR006A/B) For long PCR: TaKaRa LA Taq (TAKaRa Cat.# RR002A/B), TaKaRa LA Taq HS (TaKaRa Cat.# RR042A/B) For High Fidelity PCR: PrimeSTAR HS DNA Polymerase (TaKaRa Cat.# R010A/B) Real-time PCR reagent For SYBR Green I detection: SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa Cat.# RR041A/B) SYBR Premix Ex Taq II (Perfect Real Time) (TaKaRa Cat.# RR081A/B) For Taqan probe detection; Premix Ex Taq (Perfect Real Time) (TaKaRa Cat.# RR039A/B) IX. 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 TAKARA BIO INC. If you require licenses for other use, please call at or contact from our website at SYBR is a registered trademark of olecular Probe, Inc. Taqan is a registered trademark of Roche olecular Systems Inc. 8 TAKARA BIO INC. URL: Phone: Fax:

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