mRNA Amplification with T7 RNA Polymerase

Materials MessageAmp II aRNA Amplication Kit (Cat #1751, Ambion) 100% Ethanol RNA samples (e.g. 1 µg RNA per sample) Procedure Reverse transcription

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 H2 O 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.

Second-strand cDNA synthesis 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.cDNA purification 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 H2 O 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 H2 O.  Store at -20ºC.In vitro transcription (without biotinylation) 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 H2 O.  Vortex to mix.    Store at -20ºC.aRNA purification Preaheat Nuclease-free H2 O 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 H2 O to each cartridge.  Incubate 2 min.  Spin ~1.5 min at 10,000 xg. Store at -80ºC.