RNA Amplification using full volumes of the MessageAmp TM II aRNA Kit
A. Reverse Transcription to Synthesise First Strand cDNA
1.
Add 1  l T7 Oligo (dT) Primer to a PCR tube.
2.
Add 11  l RNA, vortex briefly and pulse spin.
3.
Incubate samples at 70  C for 10 minutes in a thermal cycler.
4.
Centrifuge samples at 4  C briefly and place on ice.
5.
Prepare Reverse Transcription Master Mix at room temperature in the order below:
Component
10X First Strand Buffer
Amount per Sample
2  l
For 12 Samples
28  l dNTP Mix 4  l 56  l
RNase Inhibitor
ArrayScript
1  l
1  l
Vortex, centrifuge briefly at 4  C and place on ice.
14  l
14  l
6.
Add 8  l of Reverse Transcription Master Mix to each sample, mix by pipetting 2-3 times, flick the tube 3-4 times and spin briefly.
7.
Incubate reactions at 42  C for 2 hours in a thermal cycler.
8.
Centrifuge briefly at 4  C and place samples on ice.

B. Second Strand cDNA Synthesis
1.
Prepare Second Strand Master Mix on ice in the order below:
Component
DEPC Water
Amount per Sample
63  l
For 12 Samples
819  l
10X Second Strand Buffer dNTP Mix
10  l
4  l
2  l
130  l
52  l
26  l DNA Polymerase
RNase H 1  l
Vortex, centrifuge briefly at 4  C and place on ice.
13  l
2.
Add 80  l Second Strand Master Mix to each sample, mix by pipetting 2-3 times, flick the tube 3-4 times and spin briefly.
3.
Incubate samples at 16  C for 2 hours in a thermal cycler, leave the lid open.
4.
Place reactions on ice.
C. cDNA Purification
1.
Add 250  l of cDNA Binding Buffer and mix by pipetting.
2.
Pipet the sample onto the centre of a cDNA Filter Cartridge.
3.
Centrifuge for 1 minute at 10,000 rpm at room temperature, discard the flow through and replace the Filter Cartridge in the wash tube.
4.
Add 500  l of Wash Buffer (ensure 100% ethanol has been added) to the Filter Cartidge.
5.
Centrifuge for 1 minute, 10,000 rpm at room temperature, discard the flow though and centrifuge for a further 1 minute at 10,000 rpm.
6.
Transfer the Filter Cartridge to a cDNA Elution Tube and elute with 20  l nuclease free water
(pre-heated to 50 – 55  C). NB. If testing cDNA concentration after the purification step, elute with 24  l water.
7.
Leave at room temperature for 2 minutes, then centrifuge for 1.5 minutes at 10,000 rpm.
8.
Re-elute with the eluate.

D. In Vitro Transcription to Synthesise aRNA
1.
Prepare an IVT Master Mix in the order below at room temperature:
Component
T7 ATP Soln (75mM)
Amount per Sample
4  l
For 12 Samples
54  l
T7 CTP Soln (75mM)
T7 GTP Soln (75mM)
4  l
4  l
4  l
54  l
54  l
54  l T7 UTP Soln (75mM)
T7 10X Reaction Buffer
T7 Enzyme Mix
4  l
4  l
54  l
54  l
Vortex and centrifuge briefly.
2.
Add 24  l IVT Master Mix to each sample, mix by pipetting 2-3 times, flick the tube 3-4 times and spin briefly.
3.
Incubate the tubes at 37  C in an oven for 14 hours.
4.
Add 60  l DEPC water to each sample and vortex to mix.
E. aRNA Purification
1.
Add 350  l of aRNA Binding Buffer to each sample.
2.
Immediately add 250  l of 100 % ethanol, mix by pipetting and transfer to an aRNA Filter
Cartridge.
3.
Centrifuge for 1 minute, 10,000 rpm and discard the flow through.
4.
Add 650  l Wash Buffer, centrifuge for 1 minute, 10,000 rpm and discard the flow through.
Centrifuge for a further 1minute, 10,000 rpm and transfer the Filter Cartridge to an aRNA
Collection Tube.
5.
Elute with 50  l nuclease free water (preheated to 50 – 55  C).
6.
Leave at room temperature for 2 minutes and then centrifuge for 1.5 minutes, 10,000 rpm.
7.
Re-elute with the eluate.
8.
Nanodrop and run on Bioanalyzer to assess RNA integrity.