RNA Amplification using half volumes of the MessageAmpTM II aRNA Kit

A. Reverse Transcription to Synthesise First Strand cDNA

  1. Add 0.5 l T7 Oligo (dT) Primer to a PCR tube.
  2. Add 5.5 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 / Amount per Sample / For 12 Samples
10X First Strand Buffer / 1 l / 14 l
dNTP Mix / 2 l / 28 l
RNase Inhibitor / 0.5 l / 7 l
ArrayScript / 0.5 l / 7 l

Vortex, centrifuge briefly at 4 C and place on ice.

  1. Add 4 l of Reverse Transcription Master Mix to each sample, mix by pipetting 2-3 times, flick the tube 3-4 times and spin briefly.
  2. Incubate reactions at 42 C for 2 hours in a thermal cycler.
  3. 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 / Amount per Sample / For 12 Samples
DEPC Water / 31.5 l / 409.5 l
10X Second Strand Buffer / 5 l / 65 l
dNTP Mix / 2 l / 26 l
DNA Polymerase / 1 l / 13 l
RNase H / 0.5 l / 6.5 l

Vortex, centrifuge briefly at 4 C and place on ice.

  1. Add 40 l Second Strand Master Mix to each sample, mix by pipetting 2-3 times, flick the tube 3-4 times and spin briefly.
  2. Incubate samples at 16 C for 2 hours in a thermal cycler, leave the lid open.
  3. Place reactions on ice.

C. cDNA Purification

  1. Add 125 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 250 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 10 l nuclease free water (pre-heated to 50 – 55 C). NB. If testing cDNA concentration after the purification step, elute with 12 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.
  9. Store samples overnight (or over the weekend) at -20 C.

D. In Vitro Transcription to Synthesise aRNA

  1. Prepare an IVT Master Mix in the order below at room temperature:

Component / Amount per Sample / For 12 Samples
T7 ATP Soln (75mM) / 2 l / 27 l
T7 CTP Soln (75mM) / 2 l / 27 l
T7 GTP Soln (75mM) / 2 l / 27 l
T7 UTP Soln (75mM) / 2 l / 27 l
T7 10X Reaction Buffer / 2 l / 27 l
T7 Enzyme Mix / 2 l / 27 l

Vortex and centrifuge briefly.

  1. Add 12 l IVT Master Mix to each sample, mix by pipetting 2-3 times, flick the tube 3-4 times and spin briefly.
  2. Incubate the tubes at 37 C in an oven for 6 hours.
  3. Add 30 l DEPC water to each sample and vortex to mix.

E. aRNA Purification

  1. Add 175 l of aRNA Binding Buffer to each sample.
  2. Immediately add 125 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 325 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.