RevoScript TM Reverse Transcriptase PreMix Kit

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1 Novel Avian Thermostable Single Polypeptide RTase RevoScript TM Reverse Transcriptase PreMix Kit Manual Ver. 1.3 Manual for RevoScript TM Reverse Transcriptase PreMix Kit for 1 st cdna Synthesis IP25083 RevoScript TM (Oligo dt 15 Primer) 48 T IP25084 RevoScript TM (Oligo dt 15 Primer) 96 T X 2 IP25085 RevoScript TM (Random Primer) 48 T IP25086 RevoScript TM (Random Primer) 96 T X 2 Issue No. ISPM intron Biotechnology, Inc.

2 Contents Product Information 2 Notice Before Use 3 General Information Storage 4 Precautions and Safety Information Product Warranty and Satisfaction Guarantee Reagents and Instruments Required 1. Description 5 2. Protocol for First-Strand cdna Synthesis 6 Manual Contents 3. Recommendations for PCR 6 4. Experimental Data 7 5. Troubleshooting Guide 9 6. Protocol Summary Related Products 11 Reverse Transcriptase In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cdna. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this. Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin Madison, and independently by David Baltimore in 1970 at MIT. The two shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco for their discovery. - Source : Wikipedia intron Biotechnology, Inc. 1

192 Tests 96 Tubes x 2 (Oligo dt 15 Primer) IP25085 48 Tests 48 Tubes (Random Primer) IP25086 192 Tests 96 Tubes x 2 (Random Primer) Components RevoScript TM RT PreMix Kit (Oligo dt 15 Primer; for 20

IP25085 / IP25086 Test tube color : Yellow Trademarks : intron, DNA-spin, DNA-midi, DNA-maxi, PCRquick-spin, MEGA-spin, MEGAquick-spin, MEGA-bead, PROBER, G- DEX, G-spin, Viral Gene-spin, easy-spin,

3 Product Information Product Cat. No. Tests Kit Contents RevoScript TM RT PreMix Kit (Oligo dt 15 Primer; for 20 μl rxn) RevoScript TM RT PreMix Kit (Random Primer; for 20 μl rxn) IP Tests 48 Tubes (Oligo dt 15 Primer) IP Tests 96 Tubes x 2 (Oligo dt 15 Primer) IP Tests 48 Tubes (Random Primer) IP Tests 96 Tubes x 2 (Random Primer) Components RevoScript TM RT PreMix Kit (Oligo dt 15 Primer; for 20 μl rxn) (Random Primer; for 20 μl rxn) RevoScript TM RTase Reaction Buffer RNase Inhibitor Primer - Oligo dt 15 Primer or Random Hexamer Stabilizer dntp Mixture RevoScript TM RT PreMix Kit (Oligo dt 15 Primer; for 20 μl rxn) Cat. No. IP25083 / IP25084 Test tube color : Transparent RevoScript TM RT PreMix Kit (Random Primer; for 20 μl rxn) Cat. No. IP25085 / IP25086 Test tube color : Yellow Trademarks : intron, DNA-spin, DNA-midi, DNA-maxi, PCRquick-spin, MEGA-spin, MEGAquick-spin, MEGA-bead, PROBER, G- DEX, G-spin, Viral Gene-spin, easy-spin, RNA-spin, easy-blue, easy-red, WEST-one, WEST-ZOL, PRO-PREP, SMART, PRO- MEASURE, Genelator, F-Detector, Broad-Way, PRO-STAIN, plug, Maxime, i-taq, i-startaq, i-max, i-starmax, RedSafe, Muta-Direct, e-myco, M-Solution, CENDORI, VeTeK, innoplex, GxN, telefaxgene, CLP and IQeasy are trademarks of intron Biotehcnology, Inc. intron kits are intended for research use only. Prior to using them for other purposes, the user must validate the system in compliance with the applicable law, directives, and regulations. The PCR process is covered by patents issued and applicable in certain countries. intron Biotechnology, Inc. does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license intron Biotechnology, Inc., all rights reserved. intron Biotechnology, Inc. 2

4 Notice Before Use 1) RNA Sample Although the RevoScript TM RT PreMix Kit has a high tolerance for some inhibitory compounds, RNA of a high purity is important for full-length, high quality cdna synthesis. This product is designed for use with 0.3 pg to 4 μg of total RNA or 0.03 pg to 400 ng of poly(a) + RNA. For the preparation of total RNA, we recommend using one of our products, either the easy-blue TM Total RNA Extraction Kit, the easy-spin TM Total RNA Extraction Kit, the easy-red TM Total RNA Extraction Kit or the easy-red TM BYF Total RNA Extraction Kit (the choice of the RNA Extraction Kit is dependent upon the species of the RNA and the source of the RNA). 2) cdna Synthesis Reaction Conditions For difficult or high GC-content templates, set the cdna synthesis temperature to 55~60. 3) Primers The cdna synthesis can be primed using random hexamers or oligo(dt) primers: Random hexamers are the most nonspecific priming method and are typically used for difficult or high GC-content RNA. Using random hexamers, all of the RNAs in a population are templates for the first-strand cdna synthesis. Oligo(dT) primers, which result in a more specific priming method, are used to hybridize to the 3 poly(a) tails, which are found in the vast majority of eukaryotic mrnas. As poly(a) + RNA constitutes approximately 1% to 2% of the total RNA, the amount and complexity of the cdna produced is considerably less than that produced with random hexamers. intron Biotechnology, Inc. 3

5 Storage Upon receipt, store the RevoScript TM RT PreMix Kit at -20 in a constant temperature freezer. If it is stored under the recommended conditions, the product will maintain its performance through the valid date that is printed on the label. Precautions and Safety Information All chemicals should be considered as potentially hazardous. When working with chemicals, always wear a suitable lab coat and disposable gloves. Only persons who are trained in laboratory techniques and are familiar with the principles of good laboratory practice should handle these products. Product Warranty and Satisfaction Guarantee All products undergo extensive quality control testing and are guaranteed to perform as described when used correctly. Any problems should be reported immediately. A satisfaction guarantee is conditional upon the customer providing the full details of the problem to intron within 60 days and returning the product to intron for examination. Reagents and Instruments Required (Not Supplied) Thermal cycler Ice RNase/DNase-free water Micro-centrifuge Vortex mixer intron Biotechnology, Inc. 4

6 1. Description The RevoScript TM RT PreMix Kit is designed for the efficient, high-yield synthesis of full-length cdna copies from either a total or poly(a) + RNA sample. The kit is optimized for a Reverse Transcriptase (RTase) reaction that can utilize a wide range RNA input amounts (from 0.3 pg to 4 μg of total RNA) and is applicable for the synthesis of cdna sized ranging from about 100 bp or less to longer than 9 kb. The RTase contained in the kit is a proprietary novel avian thermostable single polypeptide RTase derived from an avian retrovirus (Reticuloendotheliosis virus; REV). REV is a retrovirus that is immunologically, morphologically and structurally distinct from the leucosis/sarcoma group of avian retroviruses. In addition, REV has a significant phylogenetic relationship with mammalian C-type retroviruses. Because of these characteristics of REV, the RTase derived from REV contains the good qualities of both the avian retrovirus-originated RTases (more stable at higher temperatures) and the mammalian C-type retrovirus-originated RTases (single polypeptide). REV-RTase MMLV-RTase AMV-RTase Origin Reticuloendotheliosis Virus (avian) Moloney Murine Leukemia Virus Avian Myeloblastosis Virus Optimum Temperature 42~60 37~42 42~55 Structure Monomer Monomer Heterodimer The RTase of the RevoScript TM RT PreMix Kit is still active at temperatures greater than 50. Due to its good thermal stability, formation of a stable complex of primer/template/rtase is enhanced; it is desirable for the RTase to have full activity at both a relatively high temperature and the conventional reaction temperature, which can enhance sensitivity, specificity and cdna yield at a temperature range of 37~60. Specifically, it is extremely effective in the synthesis of full-length cdna from an RNA molecule with a complicated structure that could cause difficulties in template denaturation. In addition, the RTase contained in the kit has a good inherent tolerance for inhibitory compounds, which can provide an advantage when dealing with clinical samples. A half life of 260 minutes at 50 for the highest cdna yields from general RNA molecules A half life of 70 minutes at 60 for the highest cdna yields from difficult or high GC-content RNA molecules Excellent elongation ability (longer than 9 kb) Highest sensitivity (can use less than 0.3 pg of total RNA) Two kinds of kits are available: the "Oligo dt 15 Primers kit and the "Random Primers kit. Premixed all-in-one type - Ready-to-use type - Single tube format (to reduce reaction variability) - Good product shelf life (dry-formulated) - Suitable for high-throughput analysis (improved reproducibility and a rapid protocol) - Vacuum-packed to prevent the kit contents from being damaged by oxidation and hydration - Optimized for qpcr and all types of RTase reactions intron Biotechnology, Inc. 5

7 2. Protocol for First-Strand cdna Synthesis 1. Add template RNA and RNase/DNase-free water into the RevoScript TM RT PreMix tubes (Oligo dt 15 or Random Primer) to a total volume of 20 μl. Component Concentration Template RNA Total RNA 0.3 pg~4 μg poly(a) + RNA 0.03 pg~400 ng RNase/DNase-free water Total Reaction Volume To a total volume of 20 μl 20 μl 2. Briefly mix the combined reagents by vortexing, and then centrifuge briefly to collect residual liquid from the walls of the tube. To acquire reproducible data, it is important that the reaction mixture is thoroughly mixed. 3. Perform the cdna synthesis reaction as follows using a thermal cycler: Reaction Step Temperature Time cdna Synthesis min RTase Inactivation Step 95 5 min 4. Store the synthesized cdna at -20 C, or proceed directly to subsequent uses such as PCR. The appropriate amounts of cdna for PCR are described below. See 3. Recommendations for PCR. 3. Recommendations for PCR The first-strand cdna that is synthesized by the RTase reaction may be amplified directly using PCR. Optimal PCR conditions, such as the amount of cdna template and primers, may vary according to the individual PCR and must therefore be individually determined. We recommend using 10% of the RTase reaction mix (2 μl) for PCR. However, for some targets, increasing the amount of the RTase reaction mix up to 10 μl may result in enhanced PCR efficiency. Werecommend using the following DNA polymerases according to the individual amplification: i-startaq DNA Polymerase (Cat. No ) provides hot-start PCR conditions for enhanced specificity and sensitivity. It is recommended for targets of a size up to 6 kb. i-starmax II DNA Polymerase (Cat. No ) provides enhanced specificity and, fidelity as well as higher yields for long length amplification, up to 20 kb. i-pfu DNA Polymerase (Cat. No ) has a proofreading 3 to 5 exonuclease activity and provides maximum fidelity for PCR. It is recommended for targets of a size up to 9 kb. intron Biotechnology, Inc. 6

4. Experimental Data High cdna synthesis efficiency with difficult or high GC-content template 18S ribosomal RNA is a representative RNA molecule that has a highly complicated structure; reverse

8 4. Experimental Data High cdna synthesis efficiency with difficult or high GC-content template 18S ribosomal RNA is a representative RNA molecule that has a highly complicated structure; reverse transcription with this template has been considered very difficult. RevoScript TM RT retains its thermostability at temperatures greater than 50, allowing the reverse transcription to be performed at relatively high temperature. Higher temperatures can provide higher cdna synthesis efficiency with difficult or high GC-content RNA molecules than can RTase reactions performed at the conventional reaction temperature. 37 ºC 42 ºC 50 ºC 60 ºC M M M kb Fig. 1. Influence of temperature on cdna synthesis efficiency for RNA molecules with complicated secondary structures such as stem-loop configurations Total RNA was purified from human cells (SNU-1) using an easy-spin TM Total RNA Extraction Kit (intron, Cat. No ). The extracted RNA was serially diluted from 200 ng to 200 pg (10-fold serial dilution), and then firststrand cdna synthesis reactions were performed using the RevoScript RT PreMix Kit under different reaction temperature conditions. After cdna Synthesis, the 1.6 kb of DNA fragment was amplified with the RTase reaction mix using a Maxime PCR PreMix Kit (i-startaq). Lane 1: 200 ng RNA template, lane 2: 20 ng RNA template, lane 3: 2 ng RNA template, lane 4: 0.2 ng RNA template, and lane M: 1 kb DNA Ladder. Efficient full length cdna synthesis of sizes longer than 9 kb Variously sized cdnas were synthesized using either a RevoScript TM RT PreMix Kit or competitors kits. After cdna synthesis, PCRs were performed with the variously sized cdnas to compare the efficiency of the cdna synthesis. The result confirmed that the RevoScript TM RT PreMix Kit provided a higher efficiency of cdna synthesis from the RNA molecules with complicated structures or long length than did the competitors kits. A B C M M M M 9kb 4.5kb 2.7kb 1.6kb 1kb 575 bp Fig. 2. Comparison of cdna synthesis efficiency of RevoScript TM RT PreMix Kit with competitors kits To compare the efficiency of the cdna synthesis, cdnas of various sizes were synthesized using each kit, and then PCRs were performed with the synthesized cdnas using a Maxime PCR PreMix Kit (i-starmaxii). Panel A: RevoScript TM RT PreMix Kit, Panel B: RTase reaction kit of Supplier A, and Panel C: RTase reaction kit of Supplier B. Lane 1: 575 bp target gene, lane 2: 1 kb target gene, lane 3: 1.6 kb target gene, lane 4: 2.7 kb target gene, lane 5: 4.5 kb target gene, lane 6: 9 kb target gene, and lane M: 100 bp DNA Ladder. intron Biotechnology, Inc. 7

High sensitivity of cdna synthesis (as little as 0.3 pg) Human total RNA was serially diluted for the reverse transcription reaction, and the sensitivity of the cdna synthesis was tested.

9 High sensitivity of cdna synthesis (as little as 0.3 pg) Human total RNA was serially diluted for the reverse transcription reaction, and the sensitivity of the cdna synthesis was tested. The test result showed that the synthesis of cdna was possible using as little as 0.3 pg of total RNA. M bp Fig. 3. cdna synthesis sensitivity of RevoScript TM RT PreMix Kit The cdna synthesis was performed using serially diluted RNA using the RevoScript TM RT PreMix Kit. Subsequently, PCRs were performed with the synthesized cdna (187 bp) using a Maxime PCR PreMix Kit (i- StarTaq), and then the PCR mixtures were analyzed using an automatic capillary DNA analyzer. Lane M: molecular weight marker, lane 1: 1 ng RNA template, lane 2: 0.2 ng RNA template, lane 3: 40 pg RNA template, lane 4: 8 pg RNA template, lane 5: 1.6 pg RNA template, lane 6: 320 fg RNA template, lane 7: 64 fg RNA template, and lane 8: non-template control. Suitable for real-time PCR applications The cdna synthesized with the RevoScript TM RT PreMix Kit is suitable for real-time PCR applications as well as end-point PCR. The RevoScript TM RT PreMix Kit showed a superior sensitivity and a wide linear dynamic range when compared to competitors products. RevoScript RT Supplier B Supplier C Fig. 4. Suitability of RevoScript TM RT PreMix Kit for real-time PCR After cdna synthesis, the RNase P gene was amplified from the synthesized cdna with specific primers and probes using an ABI 7500 DNA analyzer. The cdna was synthesized in triplicate according to manufacturers instructions. intron Biotechnology, Inc. 8

10 5. Troubleshooting Guide This troubleshooting guide may be helpful in solving problems that may frequently arise. The scientists at intron are always happy to answer any questions you may have about the information or protocol in this manual or other molecular biology applications. Problems No or faint bands after analysis of amplified products 1) Procedural error in first-strand cdna synthesis Repeat the procedure carefully. Recommendations 2) Incorrect set-up Be sure to set-up the reaction on ice. 3) Insufficient mixing of reaction master mix during vortexing Vortex tube thoroughly. It is important that the dried material contained in each tube is sufficiently dissolved with template solution and D.W. 4) Volume of reverse-transcription reaction added to the subsequent PCR was too high Adding an excessive volume of reverse-transcription reaction to the PCR mix may reduce PCR efficiency. Generally, the volume of reverse-transcription reaction added to the subsequent PCR should not exceed 10% of the final PCR volume. 5) Pipetting error Check the pipette first to minimize pipetting error. Calibration is essential to ensure correct pipette operation. Repeat the preparation of reaction mix using a correct pipette. 6) Poor quality of RNA template Check the concentration, integrity, and purity of RNA template. Especially, in the case with the small amount of RNA template, the result of cdna synthesis may be affected by micro-amount of RNase included in sample. 7) Too high or too low RNA concentraction Measure RNA concentraction accurately to avoid too high or too low RNA concentraction. RevoScript Reverse Transcriptase Premix Kit is designed for 0.3 pg 4 μg of total RNA. It corresponds to the entire amount of RNA present including any rrna, mrna, viral RNA and carrier RNA present regardless of the primers used or cdna analyzed. In case of > 4 μg RNA, proportionally scale up the reaction to the appropriate volume. 8) RNase contamination Maintain aseptic conditions to prevent RNase contamination. Spray and wipe out the contaminent with RNase WiPER TM (intron, Cat. No ) on your experimental surface. intron Biotechnology, Inc. 9

11 Problems No or faint bands after analysis of amplified products 9) Target mrna contains strong transcriptional pauses 10) Incorrect primer concentration or primer degradation in PCR Recommendations Use random hexamers instead of oligo(dt) primer in the firststrand cdna synthesis. Increase the reaction temperature stepwise, for example, by 5 steps, up to 60. Use PCR primers closer to the 3 terminus of the target cdna Check the concentration and integrity of the primer used for PCR. If necessary, perform PCR with different primer concentrations or another primer. 11) Evaporation of reaction mix A perfect sealing is required to avoid evaporation of reaction mix during reaction. Pay attention to cap tightly. 12) Inappropriate reaction temperature Reverse-transcription reaction should be carried out at temperature lower than 65 C. Check the actual temperature of your heating block or water bath. 13) Short incubation time The standard reverse-transcription reaction requires a 60 min incubation. In rare cases, when analyzing RNAs with a very high degree of secondary structure, it may be advantageous to increase the incubation time to 2 hr. 14) Inhibitors of RTase present Remove the inhibitors by ethanol precipitation of the RNA preparation before cdna synthesis. Include a 70% (v/v) ethanol wash of the RNA pellet. Note: Example of inhibitors of RTase are sodium dodecyl sulfate (SDS), EDTA, guanidinium salts, formamide, sodium pyrophosphate, spermidine, etc. Short length of cdna products 15) Too high incubation temperature Reverse-transcription reaction should be carried out at temperature lower than 65 C. Higher temperatures than 65 C may reduce the length of cdna products. Check the actual temperature of your heating block or water bath. 16) Heat inactivation In analysis of long cdnas, heat inactivation of RevoScript TM Reverse Transcriptase is not recommended, which may cause the reduced amount of full-length cdna template resulting in a shorter PCR product than expected. intron Biotechnology, Inc. 10

12 6. Protocol Summary Reaction Mixture Preparation Adding template RNA and RNase/DNase-free water (to a total volume of 20 μl) Brief Mixing Brief mixing & centrifugation cdna Synthesis 50, 1 hr (routine sample) 60, 1 hr (difficult and high GC-content RNA) RTase Inactivation 95, 5 min PCR Amplification PCR with 2 μl of RTase reaction mix This is summary of 2. Protocols for First-Strand cdna Synthesis. 7. Related Products Product Cat. No. Size Kit Contents Maxime TM PCR PreMix Kit (i-startaq) Tests Maxime TM PCR PreMix Kit (i-starmax II) Tests Maxime TM PCR PreMix Kit (i-pfu) Tests i-startaq TM DNA Polymerase dntps Reaction buffer Gel loading buffer i-starmax TM (II) DNA Polymerase dntps Reaction buffer Gel loading buffer i-pfu DNA Polymerase dntps Reaction buffer Gel loading buffer Product Standard Hot-start Long-PCR High-fidelity Maxime TM PCR PreMix (i-taq) Maxime TM PCR PreMix (i-startaq) Maxime TM PCR PreMix (i-starmax II) Maxime TM PCR PreMix (i-pfu) intron Biotechnology, Inc. 11

#701~704, JungAng Induspia V, Sangdaewon-dong, Jungwon-gu, SeongNam-si, Gyeonggi-do, 162-120, KOREA TEL :

13 #701~704, JungAng Induspia V, Sangdaewon-dong, Jungwon-gu, SeongNam-si, Gyeonggi-do, , KOREA TEL : FAX : Issue No. ISPM intron Biotechnology, Inc.

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