COMPLEMENTARY DEOXYRIBONUCLEIC ACID (cdna) SYNTHESIS USING REVERSE TRANSCRIPTASE
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1 COMPLEMENTARY DEOXYRIBONUCLEIC ACID (cdna) SYNTHESIS USING REVERSE TRANSCRIPTASE OBJECTIVE: This Standard Operating Procedure (SOP) describes for the synthesis of from purified Ribonucleic Acid (RNA). It uses reverse transcriptase, also known as RNA-dependent Deoxyribonucleic Acid (DNA) polymerase, to transcribe single-stranded RNA into double-stranded DNA. Often cdna is made in preparation for Real-Time RT-PCR but this technique can be performed for many other downstream gene expression analyses. SCOPE: This procedure applies to all Massachusetts laboratory personnel responsible for the process of cdna synthesis using Reverse Transcriptase. RESPONSIBILITY: It is the responsibility of the Laboratory Operations Manager and Quality Assurance Officer to ensure all laboratory personnel are properly trained in and follow this SOP. SAFETY: All laboratory personnel should be in compliance with UMASS Employee Health and Safety regulations when working in the laboratory. Protective equipment, such as a lab coat and disposable gloves must be worn when working in the lab. Precautions for working with RNA: RNA is very unstable. Keep samples out of direct sunlight. NEVER VORTEX RNA. Always wear gloves while handling reagents and RNA samples. Change gloves frequently and spray gloves with 70% ethanol regularly. Work quickly and keep RNA on ice to reduce the chance of degradation by Ribonucleases (RNases.) RNases are very stable enzymes and are difficult to inactivate. Small amounts of RNases are sufficient to destroy RNA. Using RNaseZap, a solution that completely removes RNase contamination, is helpful in prepping equipment and workspace. See Section 2.1 for more details. Use proper microbiological, aseptic technique. Hands and dust particles may carry bacteria and RNases. Handle tubes as little as possible and avoid touching the inside of the lids. Keep tubes closed when not in use. Work with sterile disposable polypropylene tubes. Buy tubes that are certified to be RNase-free. Use caution in keeping them sterile once the package is opened. Keep amplified PCR products and plasmids away from RNA samples. ABBREVIATIONS AND DEFINITIONS RNA: Ribonucleic Acid RNases: Ribonucleases DNA: Deoxyribonucleic Acid 111
2 cdna: Complementary Deoxyribonucleic Acid PCR: Polymerase Chain Reaction RT-PCR: Reverse Transcription Polymerase Chain Reaction Real-Time RT-PCR: Real-Time Reverse Transcription Polymerase Chain Reaction SOP: Standard Operating Procedure UMASS: University of Massachusetts Medical School REFERENCES Mastercycler ep Instruction Manual, Eppendorf. 1. MATERIALS REQUIRED 1.1. EQUIPMENT Microcentrifuge (with adapters for 0.2 ml Tubes) Timer (Fisher S90861) Micropipettors P2 (0.2 µl to 2 µl) P20 (0.5 to 20 µl) P200 (20 to 200 µl) P1000 (200 to 1000 µl) PCR Tube Rack (Fisher ) Mastercycler ep thermal cycler (Eppendorf 5341) 1.2. SUPPLIES Kimwipes (Fisher 06666A-C) Disposable Nitrile gloves (World Wide Medical Supplies ) Sterile 0.2 ml PCR tubes (Ambion through VWR ) RNase-free Microcentrifuge Tubes (1.5 ml) (Fisher ) Sterile, Micropipettor filtered (Corning 4807, 4821, 4823, 4809) tips to fit P2, P20, P200 and P1000 Micropipettors 1.3. REAGENTS SuperScript III First-Strand (Invitrogen ) Synthesis System for RT-PCR Contents (stored at -20 C): 50 µl Oligo (dt) (50 µm) 250 µl Random Hexamers (50 ng/µl) 1 ml 10 x RT buffer 500 µl 25mM MgCl2 250 µl 0.1 M DTT 500 µl 25 mm dntp mix 50 µl SuperScript III RT (200 U/µl) 100 µl RNaseOUT (40 U/µl) 50 µl E. coli RNase H (2 U/µl) 1.2 ml DEPC-treated water 20 µl Total HeLa RNA (10 ng/µl) 112
3 25 µl Sense Control Primer (10 µm) 25 µl Antisense Control Primer (10 µm) RNaseZap Solution (Fisher NC ) Squirt Bottle of 70% ethanol (Fisher and Fisher NC ) RNase-free Water (Fisher BP24701) 2. PREPARATION 2.1 PREPARE LABORATORY BENCH AND EQUIPMENT 1. Wash hands thoroughly with soap. 2. Rinse them completely with tap water. 3. Dry hands with paper towel. 4. Put on appropriate-sized gloves. 5. Spray workspace on laboratory bench thoroughly with 70% ethanol and wipe dry with a Kimwipe. Spray workspace with RNaseZap and wipe thoroughly with a Kimwipe. Rinse with 70% ethanol again, and then dry with a Kimwipe. 6. Spray a Kimwipe with RNaseZap and wipe any micropipettors that you may be using. Wet a Kimwipe with ethanol and wipe pipettes again. Allow to dry. 7. Spray gloves with RNaseZap and spread solution all over gloves, and then spray gloves with 70% ethanol. Allow to dry. 8. Confirm that microcentrifuge is at room temperature (no cooler than 20 C). 2.2 THAW REAGENTS AND PROGRAM CYCLER 1. Remove reagents to be used from the SuperScript III First-Strand Synthesis System and thaw on ice. 2. Program Cycler as follows (if there is a saved program for reverse transcriptase, skip this step but confirm that it is correct by following Section Turn on Cycler. 4. On Start Screen, select a User, and enter login. 5. Press the NEW FOLDER function key. 6. Create a name, and click OK when complete. (Example: Reverse Transcriptase.) 7. Inside the folder create a new program by clicking on the NEW PROGRAM function key. 8. Create a name, and click OK when complete (Ex. RT1.) 9. Enter required settings. For RT 1 the program is 65 C for 5 minutes. 10. Repeat steps 7-9 for the remainder of the cycling programs needed. 2.3 CONFIRM CYCLER PROGRAMS RT1 65 C for 5 Min. RT2 25 C for 10 Min. 50 C for 50 Min. 85 C for 5 Min. RT3 37 C for 20 Min. 113
4 2.4 LABEL TUBES 1. Label three tubes for each RNA sample: First tube: +RT with the sample name (includes RNA and reverse transcriptase enzyme) Second tube: -RT (includes RNA but no reverse transcriptase enyme) Third tube: No RNA (includes reverse transcriptase but no RNA). 3. PROCEDURE 1. For each RNA sample use 1 µg of RNA per reaction. Using the concentration that was calculated in SOP CH-004 Quantification of RNA dilute the samples in RNase-free water into a labeled and sterile 0.2 µl PCR tube, so that each samples final concentration is 1 µg/µl. Prepare at least 3µl (3 µg) of diluted RNA per sample in order to have sufficient RNA for two samples and to account for pipetting errors. Note: If samples are already more dilute than 1 µg/µl, but no more dilute than 1 µg/8µl, use a higher volume of RNA and adjust the water volume in the reaction. Alternatively, if samples are more dilute than 1 µg/8 µl, use less RNA in each reaction, but all reactions must have the same amount of RNA. The steps below will need to be modified if not using 1 µl of total RNA in the next step. SuperScript III First-Strand Synthesis System for RT-PCR is optimized for 1 pg to 5µg of total RNA. 2. For each sample, prepare the RNA/primer mixtures in sterile 0.2 ml PCR tubes in the order listed, as follows: Components +RT Reaction -RT (Negative Control) Random Hexamers (Primer) 1 µl 1 µl 10 mm dntp mix 1 µl 1 µl RNase-free water 8 µl 7 µl Diluted total RNA (1 µg/µl) 1 µl 0 µl Each reaction total: 10 µl 10 µl 3. Mix briefly by pipetting up and down after the final addition of RNA to each tube. 4. Briefly centrifuge tubes. 5. Confirm that all tube lids are tightly closed. Place tubes in cycler and close lid. 6. Select the RT1 program (65 C for 5 Min.) and select Start Run. 7. While the RT1 program is running, prepare cdna Synthesis Mix for each sample. 8. Using the chart below, determine how many reactions are needed. Remember to add an additional sample to account for pipetting error. 9. Add the following reagents to an RNase-free microcentrifuge tube labeled cdna Synthesis Mix in the order they are listed for +RT and No RNA reactions: 114
5 Component 1 Reaction 10 Reactions 10 x RT buffer 2 µl 20 µl 25mM MgCl 2 4 µl 40 µl 0.1 M DTT 2 µl 20 µl RNaseOUT (40 U/µl)1 µl 10 µl 50 µl SuperScript III RT (200 U/µl)1µl 10 µl For RT reactions prepare the following: Component 1 Reaction 10 Reactions 10 x RT buffer 2 µl 20 µl 25 mm MgCl 2 4 µl 40 µl 0.1 M DTT 2 µl 20 µl RNaseOUT (40U/µl)1 µl 10 µl Water 1µl 1 µl 10. Once the run is complete, remove each tube carefully from the cycler. Place samples on ice for at least 1 minute. 11. Add 10 µl of cdna Synthesis Mix to each sample. Mix gently by pipetting up and down. 12. Briefly centrifuge tubes. 13. Confirm that all tube lids are tightly closed. Place tubes in cycler and close lid. 14. Select the RT2 program (25 C for 10 Min., 50 C for 50 Min., 85 C for 5 Min.) and select Start Run. 15. Once the run is complete, remove each tube carefully from the cycler. Place samples on ice. 16. Collect the reactions with a brief centrifugation. Add 1 µl of E. coli RNase H (2 U/µl) to each tube. Mix gently by pipetting up and down. 17. Confirm that all tube lids are tightly closed. Place tubes in cycler and close lid. 18. Select the RT3 program (37 C for 20 minutes) and select Start Run. 19. Once the run is complete, remove each tube carefully from the cycler. Place samples on ice. Note: cdna synthesis reaction can be kept on ice if used immediately, or it can be stored in a -80 C freezer. 115
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