Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol (5184-3523).

Fluorescent cRNA Synthesis Procedure

cDNA Synthesis From Total RNA

(Time required: ~3 hours)

1. Add 50 to 500 ng total RNA in a volume of 10.3 µL □

or less. The total concentration should be at least

5 ng/µL.

2. Add 1.2 or 5 µL of T7 Promoter Primer (from kit).□

3. Use nuclease-free water to bring the total reaction □

volume to 11.5 µL.

4. Incubate the reaction at 65°C in a heating block for □

10 minutes.

5. Place the reactions on ice and incubate for 5 minutes.□

6. Immediately—prior to use, gently mix the following □

components by pipetting, in the order indicated, at room temperature:

Be sure to always make a mix for 10% (round up to whole) reactions more or at least 1.

Pre-warm the 5X First Strand Buffer by incubating the

vial in a 65°C waterbath for 3-4 minutes.

Vortex briefly and spin the tube briefly in a microfuge.

7. To each sample tube, add 8.5 µL cDNA Master Mix.□

8. Incubate samples at 40°C in a circulating water bath□

for 2 hours.

9. Move samples to a heating block or water bath set to□

65°C and incubate for 15 minutes.

10. Move samples to ice. Incubate on ice for 5 minutes.□

11. Spin samples briefly in a microcentrifuge. □

Fluorescent cRNA Synthesis: in vitro transcription and incorporation of cyanine 3- or cyanine 5-CTP

(Time required: 2.5 hours)

12. Immediately, prior to use, gently mix the following □

components by pipetting, in the order indicated, at

room temperature:

Be sure to always make a mix for 10% (round up to whole) reactions more or at least 1.

Pre-warm the 50% PEG solution by incubating the vial

in a 40°C waterbath for 1 minute. Vortex briefly and

spin the tube briefly in a microfuge.

13. To each sample tube, add 60.0 µL of Transcription □

Master Mix. Gently mix by pipetting.

# / Name / Label with
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14. Incubate samples in a circulating water bath at 40°C □

for 2 hours.

Purification of Amplified cRNA (Machery-Nagel NucleoSpin RNA II kit)

15. Add 20 µL of nuclease free-water to your cRNA □

sample

16. Add 350 µL of Buffer RA1 and vortex to mix.□

17. Add 350 µL of ethanol (70%) and mix □

by vortexing.

18. Transfer 800 µL of cRNA sample to an nucleospin RNA II □

column.

Centrifuge the sample for 30 seconds at 11.000 RPM.

Place the column in a new collection tube.

19.Add 200 µL of buffer RA2 to the nucleospin RNA II column. □ Centrifuge the sample for 30 seconds at 11.000 RPM.

Place the column in a new collection tube.

20. Add 600 µL of buffer RA3 to the nucleospin RNA II column.□

Centrifuge the sample for 30 seconds at 11.000 RPM.

Discard flowtrough and place column back into the collecting tube.

21. Add 250 µL of buffer RA3 to the nucleospin RNA II column.□

Centrifuge the sample for 2 min at 13.000 RPM to dry the membrane competely. Place the column into a nucleasefree 1,5 ml microcentrifuge tube.

22.Elute the RNA in 60 µL nuclease free H2O and □ centrifuge at 13.000 RPM for 1 min.

23. Add the eluate from step 22 again directly onto the □

nucleospin RNA II column.

Centrifuge at 13.000 RPM for 1 min.

1. Perform wavescan on nanodrop to determine labelling efficiency and amplification.

Save the report on disk.

2.If requested evaluate labelled sample on an Agilent 2100 bioanalyzer.

Overview Concentrations and Hybridisation

# / Name / Label / Conc. µg/ml / µg on array / µl required
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Cy5 / Cy3 / Slide barcode / Batch number
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Materiallist.

Materials for general purposes.

  • sterile nuclease-free reaction tubes (0,5 and 1,5 ml)
  • nuclease-free water
  • ice
  • Microcentrifuge
  • Incubator (waterbath or PCR machine 65ºC and 40ºC)
  • vortex
  • microplate centrifuge
  • Magnetic stir plate
  • Speedvac
  • Nanodrop
  • Bioanalyzer

cDNA Synthesis From Total RNA

  • 50 to 500 ng total RNA

Low input fluorescent amplification kit (Agilent 5184-3523):

cDNA Master Mix

  • T7 Promoter Primer
  • 5X First Strand Buffer
  • 0,1 M DTT
  • 10 mM dNTP mix
  • MMLV RT
  • RNaseOUT

Fluorescent cRNA Synthesis: in vitro transcription and incorporation of cyanine 3- or cyanine 5-CTP

Low input fluorescent amplification kit (Agilent 5184-3523):

Transcription Master Mix

  • 4X Transcription Buffer
  • 0,1 M DTT
  • NTP Mix
  • 50% PEG
  • RNaseOUT
  • Inorganic Pyrophosphatase
  • T7 RNA Polymerase
  • Cy3-CTP (Perkin-Elmer NEL580)
  • Cy5-CTP (Perkin-Elmer NEL581)

Purification of Amplified cRNA (RNeasy kit)

  • Buffer RLT
  • ethanol (96-100% purity)
  • RNeasy mini column
  • buffer RPE

Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol1

13-4-2019