TargetAmp 1-Round aRNA Amplification Kit 103 RNA Amplification Procedure A. B. C. D. First-strand cDNA Synthesis Second-strand cDNA Synthesis In Vitro Transcription of aRNA aRNA Purification A. First-strand cDNA Synthesis Required for Step A: TargetAmp T7-Oligo (dT) Primer A TargetAmp Reverse Transcriptase PreMix-SS Dithiothreitol (DTT) RNase-Free Water Superscript III Reverse Transcriptase (200 U/μl) (Red Tube) (Red Tube) (Colorless Tube) (Colorless Tube) (Not Provided in Kit) Incubations Performed in Step A: 50 ºC and 65 ºC. If the thermocycler is used make sure the lid function is also set at 50 ºC and 65 ºC in each incubation step. 1. Anneal the TargetAmp T7-Oligo (dT) Primer A to the RNA sample. For best results use no more than 500 ng of RNA for each reaction. Perform the reaction in 0.2 mL tubes (this will reduce evaporation during incubation). x μl RNase-Free Water x μl Total RNA sample (500ng) 1 μl TargetAmp T7-Oligo (dT) Primer A 3 μl Total reaction volume 2. 3. 4. 5. Gently mix the reaction by flicking and spin down the contents before incubating. Incubate at 65 ºC for 5 minutes. Chill on ice for 1 minute Prepare the 1st-strand cDNA Synthesis Master Mix For each 1st-strand cDNA synthesis reaction, combine on ice: 1.50 μl TargetAmp Reverse Transcription PreMix-SS 0.25 μl DTT 0.25 μl Superscript III Reverse Transcriptase (200 U/μl) 2.00 μl Total reaction volume 6. Gently mix the 1st-strand cDNA Synthesis Master Mix and add 2 μl to each reaction. 7. Gently mix the reactions and incubate at 50 ºC for 30 minutes. B. Second-strand cDNA Synthesis Required for Step B: TargetAmp DNA Polymerase PreMix-SS 1 TargetAmp DNA Polymerase SS-1 (Red Tube) (Red Tube) Incubations Performed in Step B: 65 ºC and 80 ºC. 1. Prepare the 2nd-Strand cDNA Synthesis Master Mix. For each 2nd-strand cDNA synthesis reaction, combine on ice: 4.5 μl TargetAmp DNA Polymerase PreMix-SS 1 0.5 μl TargetAmp DNA Polymerase 5.0 μl Total reaction volume 2. 3. 4. 5. 6. Gently mix the 2nd-Strand cDNA Synthesis Master Mix and add 5 μl to each reaction. Gently mix the reactions and incubate at 65 ºC for 10 minutes. Centrifuge down the contents. Incubate at 80 ºC for 3 minutes. Chill on ice Note: If desired, the reactions may be frozen and stored overnight at -20 ºC. C. In Vitro Transcription of aRNA Required for Step C: TargetAmp T7 RNA Polymerase TargetAmp T7 Transcription Buffer 100 mM ATP 100 mM CTP 100 mM GTP 100 mM UTP Dithiothreitol RNase-Free Water RNase-Free DNase I (Green Tube) (Green Tube) (Green Tube) (Green Tube) (Green Tube) (Green Tube) (Colorless Tube) (Colorless Tube) (Green Tube) Incubations Performed in Step A: 37 ºC and 40 ºC 1. Warm the TargetAmp T7 RNA Polymerase to room temperature. 2. Warm the TargetAmp T7 Transcription Buffer and RNase-Free water at 37 ºC for 5 minutes. Mix the buffer thoroughly to dissolve any precipitates and keep it at room temperature. 3. Prepare the In Vitro Transcription Master Mix. Important: Add reagents in order. Mix RNase-Free Water and TargetAmp T7 Transcription Buffer before adding the rest of the reagents. For each in vitro transcription reaction, combine at room temperature: 13.6 μl RNase-Free Water 4.0 μl TargetAmp T7 Transcription Buffer 3.6 μl ATP 3.6 μl CTP 3.6 μl GTP 3.6 μl UTP 4.0 μl DTT 4.0 μl TargetAmp T7 RNA Polymerase 40.0 μl Total reaction Volume 4. Gently mix the In Vitro Transcription Master Mix and add 40 μl to each reaction. 5. Gently mix the reactions and incubate at 42 ºC for 4 hours. For optimal yield and quality do not exceed 4 hours of incubation. 6. Add 2 μl of RNase-Free DNase I to each reaction. 7. Gently mix and incubate at 37 ºC for 15 minutes. D. aRNA Purification Required for Step D: Qiagen RNeasy Mini Kit RNase-Free Water (use the water provided by the Qiagen RNeasy Mini Kit) RLT/β-Mercaptoethanol Solution (RLT provided in kit, β-ME not provided in kit) RPE Solution (provided in kit) 100% Ethanol 80% Ethanol 1.7 mL centrifuge tubes Pink spin columns (provided in kit) 1. For each sample prepare 350 μl RLT/β-ME Solution. Make the solution under the fume hood. To 1.0 mL Buffer RLT add 10 μl of β-ME. 2. For each sample prepare 650 μl RPE Solution. The RPE Solution in the kit should be diluted 1 volume RPE to 4 volumes 100% Ethanol. Check to see if the solution bottle is marked as already being diluted 1:4. If you have to dilute a new solution, make sure you mark the bottle as being diluted. 3. Transfer each sample to 1.7 mL centrifuge tubes. 4. To each sample add: 48 μl RNase-Free Water 350 μl RLT/β-ME Solution 250 μl 100% 5. Apply each sample to the pink spin column in a 2 mL collection tube (provided in kit). Centrifuge at >8,000 x g for 20 seconds. Discard the flow-through 6. Apply 650 μl RPE Solution onto each column. Centrifuge at >8,000 x g for 20 seconds. Discard the flow-through 7. Apply 650 μl 80% Ethanol onto each column. Centrifuge at >8,000 x g for 20 seconds. Discard the flow-through 8. Transfer the column to a new collection tube (provided in kit). Centrifuge at max speed for 5 minutes. Keep the spin column and discard the collection tube. 9. Transfer the spin column to a 1.7 mL centrifuge tube. 10. Apply 25 μl RNase-Free Water directly onto the center of the silica-gel membrane of the spin column. Wait for 2 minutes. Centrifuge at full speed for 1 minute. 11. Repeat step 10 in the same 1.7 mL collection tube. 12. The reactions can now be stored at -80 ºC.