Protocol MessageAmp II and coupling monoreactive dyes.
Protocol MessageAmp aRNA Kit.
Array Controls.
Mix / Spike / Amount Spike (µl) / Amount TE buffer (µl) / Concentration (ng/µl) / Amount Mix A t/m D (µl)A / 1 / 1 / 9 / 10
B / 3 / 1 / 9 / 10
C / 5 / 1 / 9 / 10
D / 7 / 1 / 9 / 10
E / 2 / 1 / 99 / 1
F / 4 / 1 / 99 / 1
G / 6 / 1 / 99 / 1
H / 8 / 1 / 99 / 1
************** / ************** / ************** / ************** / ************** / **************
I / 1 / - / 27 / 1 / 3 (A)
J / 3 / - / 27 / 1 / 3 (B)
K / 5 / - / 27 / 1 / 3 (C)
L / 7 / - / 27 / 1 / 3 (D)
10 ng/µl RNA Spikes / 1 ng/µl RNA Spikes
Odd numbered / Even numbered / Odd numbered
A / B / C / D / E / F / G / H / I / J / K / L / TE buffer
Cy5 Odd / 5 µl / 2,5 µl / 5 µl / 2 µl / 85,5 µl
Cy5 Even / 10 µl / 10 µl / 10 µl / 2 µl / 68 µl
Cy3 odd / 5 µl / 5 µl / 20 µl / 20 µl / 50 µl
Cy3 even / 1 µl / 2 µl / 4 µl / 2 µl / 91 µl
1. Add 10 µl Cy5 even at 10 µl Cy5 odd (Stock solution A).
2. Add 10 µl Cy3 even at 10 µl Cy3 odd (Stock solution B).
3. Dilute this 5 times for a 1 µg labeling.
4. Use 2 µl for one labeling.
First Round Amplification.
Reverse Transcriptation to Synthesize First Strand cDNA.
1. 1 µg (at least 500 ng) Total RNA (max. 9 µl).
2. Add 2 µl Spike controls → cy5 (A) for cy5 labeling and cy3 (B) for cy3 labeling.
3. Add 1 µl of T7 Oligo(dT) primer.
4. Add DEPC to a final volume of 12 µl.
5. Incubate 10 min. at 70°C.
6. Centrifuge briefly and place the samples on ice..
7. Add the following mix:
7. Resuspend the mix and centrifuge briefly.
8. Add 8 µl of the mix to each sample quickly at 42°C.
9. Incubate at 42°C for 2 hr.
10. Centrifuge the samples briefly.
11. Place the samples on ice. `
Second Strand cDNA Synthesis.
1. Add the second strand cDNA mix to the samples at RT:
2. Mix the samples and incubate for 2 hr at 16°C.
cDNA purification.
Before beginning the cDNA purification, preheat the nuclease-free water
to 50°C for at least 10 minutes.
1. Add 250 µl of cDNA Binding Buffer to each sample and resuspend.
2. Pipet this on the filter.
3. Centrifuge at 10000 rpm for 1 min.
4. Discard the flow-through.
5. Apply 500 µl cDNA Wash Buffer to each cDNA Filter Cartridge.
6. Centrifuge at 10000 rpm for 1 min.
7. Discard the flow-through.
8. Spin the cDNA Filter cartrigdge an additional minute.
9. Transfer cDNA Filter to a cDNA Elution Tube.
10. To the center of the filter pipet 18 µl of the pre-heated nuclease-free water.
11. Incubate at RT for 2 min. and then centrifuge for 1 min at 13000 rpm.
In Vitro Transcription to Synthesize aRNA.
1. Mix de following reactionmix with ±16 µl doublestranded cDNA template.
1. Incubate at 37°C for 16 hours and then put it to 4°C.
In a incubator overnight (PCR-machine).
2. Add 10 µl Nuclease-free water to stop the reaction.
3. Purify the cRNA.
aRNA purification.
Before beginning the cRNA purification, use the preheated nuclease-free water
to 50°C.
1. Put 175 µl of aRNA Binding Buffer with the sample and resuspend.
2. Add 125 µl 100% EtOH and resuspend.
3. Pipet this 350 µl on the filter.
4. Centrifuge at 10000 rpm for 1 min.
5. Discard the flow-through.
6. Apply 650 µl cRNA Wash Buffer to each aRNA Filter Cartridge.
7. Centrifuge at 10000 rpm for 1 min.
8. Discard the flow-through.
9. Spin the aRNA Filter cartridge an additional minute.
10. Transfer aRNA Filter to a aRNA Elution Tube.
11. To the center of the filter pipet 50 µl of the pre-heated nuclease-free water.
12. Incubate at RT for 2 min. and then centrifuge for 1 min at 13000 rpm.
Labeling with amine-reactive reagent.
1. Take 3 - 5 µg RNA resolved in a volume of 3,33 µl nuclease-free water.
2. Add 5 µl dry DMSO to of the amine-modified cRNA.
3. Add 1,66 µl 0,3 M Na-carbonate buffer (pH 9,0; check pH regulary)
4. Immediately transfer the mixture to the aliquotted dyes.
5. Resuspend the dye in the mixture and incubate 1 hour at RT in the dark.
6. Add 4,5 µl hydroxylamine and leave 15 min at RT in the dark.
7. Clean cRNA with te microcon YM-30 method (see standard protocol) or.
Clean cRNA with the RNeasy column method (see protocol RNeasy).
Clean the samples using the Macherey-Nagel RNAII kit.
1. Add 29 µl DEPC to the sample until it is 50 .
2. Add 175 µl RAI buffer and mix.
3. Add 175 µl ethanol 70% and mix by pipetting.
4. Put the sample on the column.
5. Centrifuge 1 min. at 11.000 rpm and remove the eluat.
6. Add 200 µl RAII buffer and centrifuge 1 min at 11.000 rpm.
7. Remove the eluat.
8. Add 600 µl RAIII buffer and centrifuge 1 min at 11.000 rpm.
9. Add 250 µl RAIII buffer and centrifuge 2 min at 13.000 rpm.
10. Remove the eluat.
11. Elute the sample by pipetting 50 µl DEPC water (preheated 50°C) on the column.
12. Incubate for 2 min.
13. Centrifuge 1 min at 13.000 rpm.
Quantitating aRNA Products
1. Perform wavescan on nanodrop to determine labelling efficiency.
2. If requested evaluate labelled sample on an Agilent 2100 bioanalyzer.
Hybridizationtabel.
Arraytabel.