mRNA Amplification with T7 RNA Polymerase
MessageAmp II aRNA Amplication Kit (Cat #1751, Ambion)
RNA samples (e.g. 1 µg RNA per sample)
Second-strand cDNA synthesis
- Verify that EtOH (24 mL) has been added to the Wash Buffer.
- Turn on a 42ºC oven and set PCR machine to hold at 70ºC.
- In an Eppendorf tube add:
1 µg Total RNA
1 µL T7 oligo(dT) primer
q.s. to 12 µL with Nuclease-free H2O
- Incubate 10 min. at 70ºC. Spin briefly to pull down any condensation. Place on ice.
- Prepare RT-Master Mix (per sample):
2 µL 10x 1st strand buffer
4 µL dNTP mix
1 µL RNase inhibitor
1 µL ArrayScript
- Add 8 µL RT-Master Mix to each RNA sample. Incubate at 42ºC for 2 hrs. Place on ice.
- Set PCR machine to hold at 16ºC.
- On ice prepare 2nd Strand Master Mix (per sample):
63 µL Nuclease-free H2O
10 µL 10x 2nd strand buffer
4 µL dNTP mix
2 µL DNA polymerase
1 µL RNase H
- Vortex master mix briefly, pull down by spinning briefly and place on ice.
- Add 80 µL 2nd Strand Master Mix to each RNA sample. Mix by pipetting, flicking and spin briefly to pull down.
- Incubate RNA at 16ºC x 2 hrs on PCR machine. Do not close lid. Place on ice or freeze o.n.
In vitro transcription (without biotinylation)
- Preheat nuclease-free H2O to 55ºC.
- Add 250 µL of cDNA Binding Buffer to each sample. Mix by pipetting and flicking, and spin briefly to pull down.
- Load sample onto a cDNA Filter Cartridge in a wash tube. Spin 1 min. at 10,000 xg. Discard flow-through.
- Add 500 µL Wash Buffer. Spin 1 min. at 10,000 xg. Discard flow-through.
- Transfer cartridge to a cDNA Elution Tube.
- Add 10 µL of 55ºC Nuclease Free H2O to the center of the cartridge. Incubate 2 min, then spin 1.5 min. at 10,000 xg.
- Repeat elution in the same tube with another 10 µL of 55ºC H2O.
- Store at -20ºC.
- Heat oven to 37ºC.
- Prepare IVT Master Mix (per sample):
4 µL T7 ATP Solution
4 µL T7 CTP Solution
4 µL T7 GTP Solution
4 µL T7 UTP Solution
4 µL T7 10x Reaction Buffer
4 µL T7 Enzyme Mix
- Add 24 µL of IVT Master Mix to each sample. Incubate at 37ºC for 4hrs - 14 hrs.
- Stop the reaction by adding 60 µL of Nuclease-free H2O. Vortex to mix. Store at -20ºC.
- Preaheat Nuclease-free H2O to 55ºC.
- Label aRNA Filter Cartridges and place in aRNA Collection Tubes.
- Add 350 µL of aRNA Binding Buffer to each aRNA sample.
- Immediately add 250 µL absolute EtOH to each sample. Mix by pipetting and transfer to aRNA Filter cartridge.
- Spin ~1 min. at 10,000 xg. Discard flow-through.
- Add 650 µL Wash Buffer to each sample. Spin ~1 min. at 10,000 xg. Discard flow-through.
- Transfer cartridges to fresh aRNA Collection Tubes.
- Add 100 µL of 55ºC Nuclease-free H2O to each cartridge. Incubate 2 min. Spin ~1.5 min at 10,000 xg.
- Store at -80ºC.