T7-Based RNA Amplification Protocol (in progress)

T7-Based RNA Amplification Protocol (in progress) Jacqueline Ann Lopez (modifications) Amy Cash & Justen Andrews INTRODUCTION T7 RNA Amplification, a technique originally developed in the laboratory of James Eberwine (Van Gelder et al. 1990), allows for linear amplification of mrna from limited samples. This technique amplifies very small RNA samples in order to produce enough material for microarray hybridizations. The final product is amplified, antisense RNA (arna). The protocol described here was adapted from Ambion s MessageAmp II Amplification kit and Klebes et al. (2002) Genome Biology 3. Depending on the starting amount of total RNA, input recommendations are as follows: Starting Material No. of Round Estimated amplification 100 ng - 1 ug total RNA 1 round ~10 3 amplification MATERIALS Item/ Description Company Cat. No. Method Rneasy Mini Kit (50 reactions) Qiagen 74104 arna purification Rneasy Mini Kit (250 reactions) Qiagen 74106 Agilent RNA 6000 Nano Complete Kit with s and Chips (25 chips) Oligo (dt)-t7 Primer (100ng/µl) Agilent 5067-1511 IDT Custom Order dntp Set (100 mm; 4x250µl each) Bioline BIO-39025 SuperScript II (10, 000U; 200U/µl) with 5X First Strand buffer (500 µl) and 0.1M DTT (500 µl) Invitrogen 18064-014 RNASEOUT (5,000U; 40U/µl) Invitrogen 10777-019 5X Second Strand Buffer (500 µl) Invitrogen 10812-014 DNA Polymerase I (1,000U; 10U/µl) Invitrogen 18010-025 Rnase H (120U) Invitrogen 18021-071 MiniElute PCR Clean Up Kit (50 reactions) Qiagen 28204 T7 MegaScript Kit (40 reactions) Ambion 1334 arna purification arna analysis 1 st Round FS 1 st Round FS & SS 1 st Round FS 1 st Round FS 1 st Round SS 1 st Round SS 1 st Round SS ds-cdna purification 1 st Round RNA amplification Nuclease-free water ---- ---- ---- Nuclease-free micro-centrifuge tubes ---- ---- ----

(0.2 ml; 0.5 ml; 1.5 ml) Table top micro-centrifuge (max. 16,000 rcf) ---- ---- ---- Thermocylcer ---- ---- ---- Nuclease-free Pipet Tips (P2, P20, P200, P1000) ---- ---- ---- Pipetors (P2, P20, P1000) FS = First Strand cdna synthesis; SS = Second Strand cdna synthesis PROCEDURE Reverse Transcription: First Round Before you start: 1. Prepare working stock of 10mM dntp mix from the 100mM stock dntp set a. Mix equal amounts of ATP, CTP, GTP, TTP (e.i. 10µl of each = 40µl) b. Add enough water for 1:10 dilution (e.i. 40 µl dntps: 400 µl final volume) 2. Dilute/Concentrate total RNA samples on ice: a. 1 ug of Total RNA in a final volume of 4.0 µl [final conc. of 250ng/µl] Method: First Strand cdna Synthesis Note: All plastic ware needs to be RNase-free/DNase-free 1. Thaw the following reagents on ice: b. total RNA (250 ng/µl) c. Oligo (dt)-t7 Primer (100ng/µl) d. 5X First Strand Buffer e. 10mM dntp mix 3. The following amounts are for a single reaction of first strand reverse transcription for single strand cdna: Total RNA (250ng/µl) 4.0 Oligo (dt)-t7 Primer (100ng/µl) 1.0 Total volume 5.0 a. Mix gently and centrifuge briefly to collect reaction mixture at bottom of tube. b. Incubate at 65 C for 10 minutes to anneal primer. c. Place on ice immediately for 3 min to stop reaction. d. Centrifuge briefly to collect reaction mix and then place on ice. 4. The following amounts are for a single reaction of first strand reverse transcription for single strand cdna: 5X First Strand Buffer 2.0 0.1M DTT 1.0

10mM dntp mix 1.0 RnaseOUT (40U/µl) 1.0 Superscript II (200U/µl) 1.0 Total Volume 6.0 a. For convenience, prepare a master mix for the number of reactions. Vortex briefly to mix and centrifuge briefly to collect the reaction mixture. b. Add 6.0 µl of master mix to each sample. c. Mix gently by pipetting. Centrifuge briefly to collect reaction mixture at bottom of tube. d. Incubate at 42 C for 2 hours and then at 65 C for 10 minutes to stop reaction. e. Place samples on ice. Proceed to second strand cdna synthesis Method: Second Strand cdna Synthesis 1. Thaw the following reagents on ice: a. 5X Second Strand Buffer b. 10 mm dntp mix 2. The following amounts are for a single reaction of second strand reverse transcription for double stranded cdna: Nuclease-free water 45.0 5X Second Strand Buffer 2.0 10mM dntp mix 1.0 DNA Polymerase (10U/µl) 2.0 RNase H (2U/µl) 1.0 Total Volume 64.5 a. For convenience, prepare a master mix for the number of reactions. Vortex briefly to mix and centrifuge briefly to collect the reaction mixture. b. Add 64.5 µl of master mix to each sample. Mix gently by pipetting. Centrifuge briefly to collect reaction mixture at bottom of tube. c. Incubate at 16 C for 2 hours. Note: Ideally these reactions should be incubated in a thermal cycler with a lid temperature that matches the block temperature. However, most machines do not have this feature so perform these reactions with the heated lid turned off. Also, it is important to cool the thermal cycler block to 16 C before adding the tubes. 3. Place samples on ice. Proceed to double stranded cdna purification cdna Purification with QIAGEN MiniElute PCR Purification Kit 1. Add 5 volumes of Buffer PBI to 1 volume of ds-cdna sample. a. Maximum Binding Capacity: 5 µg b. Maximum Loading Volume: 800 µl 2. Mix well and check that the color of the mixture is yellow. (If color is orange or violet, add 10 µl 3M sodium acetate, ph 5, and mix. The color will turn to yellow.) 3. Pipet mixture onto MiniElute column placed in a 2 ml collection tube. 4. Centrifuge for 1 min at 10,000 x g (12,000 rcf). 5. Discard flow-through and replace filter into collection tube.

6. Add 750 µl of Buffer PE to each column. 7. Centrifuge for 1 min at 10,000 x g (12,000 rcf). 8. Discard flow-through. 9. Centrifuge for an additional minute at maximum speed (16,000 rcf) to remove trace amounts of Buffer PE. a. Ethanol carryover or contamination will significantly reduce the in vitro reaction. 10. Transfer MiniElute column to a clean 1.5 ml microcentrifuge tube and discard the collection tube. 11. Add 10 µl of Buffer EB to the center of the silica-gel membrane. 12. Incubate at room temperature for 1 min. 13. Centrifuge for 1 min at 10,000 x g (12,000). 14. The elutate now contains the purified double-stranded cdna (~9 µl). 15. Quantify cdna synthesis using Nanodrop to determine the yield. 16. Add 8 µl RNase-free H 2 O to bring volume up to 16 µl. In Vitro Transcription (Ambion MEGAscript kit) Note: This reaction is currently set-up as a 40 µl IVT reaction. It would save on reagent costs to scale this down to a 20 µl reaction volume but I have not tested this yet. 1. Thaw reagents the following reactions: a. Enzyme Mix ON ICE b. Ribonucleotides ON ICE c. 10X Reaction Buffer ROOM TEMPERATURE i. The spermidine in the 10X Reaction buffer can come out of solution and co-precipitate the DNA if the reaction is assembled on ice. 2. Mix equal volumes of the 4 ribonucleotides together to create a NTP mix. 3. At room temperature, prepare an In Vitro Transcription Master Mix in a nuclease-free tube: NTP mix 16 10X Reaction Buffer 4 Enzyme Mix 4 Total Volume 24 µl 4. Mix well and centrifuge briefly. 5. Add 24 µl of master mix to each double-stranded cdna sample. 6. Mix well and centrifuge briefly. 7. Incubate at 37 C for 4 hrs. Note: We recommend that IVT reactions be performed for no longer than 4 hrs as arna degradation can occur with longer incubations. 8. Remove cdna: Add 1 µl of TURBO DNase to each IVT reaction. Mix well. Incubate at 37 C for 15 min. 9. At this point, IVT reactions can be kept at a 4 C hold overnight in the thermal cycler or taken out and stored at -20 C. arna Purification (Qiagen RNeasy Kit with slight modifications) 1. Before using the kit for the first time, prepare Buffer RPE by adding 44 ml 100% ethanol. Mix well and check the box on the label to indicate the ethanol was added. 2. Bring sample volume up to 100 µl by adding 60 µl nuclease-free H 2 O. 3. Add 350 µl of Buffer RLT to each sample and mix well. 4. Add 250 µl of 100% ethanol to each sample.

5. Mix well but do not centrifuge. 6. Immediately pipet the mixture (700 µl) onto an RNeasy column inserted in a collection tube. 7. Centrifuge for 30 sec at 10,000 x g (10,000 rcf). 8. Discard flow-through and replace filter into collection tube. 9. Add 500 µl of Buffer RPE to each filter. 10. Centrifuge for 30 sec at 10,000 x g (10,000 rcf) and discard flow-through. 11. Add 650 µl of 80% ethanol. 12. Centrifuge for 2 min at 10,000 x g (10,000 rcf) to remove trace amounts of Buffer RPE/80% Ethanol. 13. Transfer RNeasy column to a 1.5 ml microcentrifuge tube. Save collection tube for KREAtech labeling purification step. 14. Add 30 µl of RNase-free H 2 O to the center of the filter. 15. Incubate at room temperature for 1 min. 16. Centrifuge for 1 min at 10,000 x g (10,000 rcf). 17. Repeat with a second aliquot of 50 µl RNase-free H 2 O 18. The elutate now contains the purified arna. arna Quantification Assessment: Determine concentration 1. Launch Nanodrop software to start application and adjust setting for RNA quantification. Select Nucleic Acid. 2. A prompt will appear. 3. Buff top and bottom pedestal. Apply pressure gently and stroking 10 times with Kimwipe. 4. Load 1.3 μl of nuclease-free water to pedestal. Lower arm and click OK. 5. Select Sample Type from drop-down menu. Sample type: RNA 40 Extinction coefficient = 40 6. Buff pedestal both top and bottom of pedestal after every measurement. 7. To BLANK, apply to the pedestal 1.3 μl of elution buffer in which arna is dissolved. If water was used, the blank measurement is done with the same water sample using for the elution during arna purification. If another elute buffer was used, that is the buffer used to calibrate the instrument. 8. Click BLANK to measure and calibrate the instrument. Instrument is calibrated with the solvent used to elute the arna from the RNeasy mini column during purification. 9. Instrument is now ready to measure concentration of sample. Be sure to enter a Sample Name for each measurement and/or sample. 10. To quantify your sample, apply 1.3 μl of sample to pedestal and click Measure. Min. phenolate ion contamination: 260/230 >2.1

Min. protein contamination: 260/280 > 1.9 arna Qualitative Assessment: Bio Analyzer It is likewise important to guard against sources of dust and nucleases. We recommend using barrier pipette tips, and filtering all solutions (except those that are purchased as sterile and nuclease-free). Wipe down all electrophoresis equipment, pipettors and bench with RNase Zap as instructed by manufacturer. 1. Prepare arna sample (vol = 1.5 μl) to a final concentration of 250 ng/μl. Concentrate 375 ng arna to a volume of 1.5 μl in a 0.2 ml micro centrifuge tube. Mix by gently pipetting. Centrifuge briefly to collect the contents Keep sample on ice. 2. Set up Bioanalyzer software and adjust settings for mrna quantification according to manufacture s instructions. Follow manufacturer s instructions to assess the quality of the arna. Dilute the sample to a concentration of 250 ng/ μl. Over loading the chip with arna leads to inaccurate assay results. 3. Assess quality of arna from electrophrograms of arna amplification. Example: Electrophrogram and gel image of arna; Phototactic-fish control replicate #1.