Meyerowitz lab, Caltech Last update: Feb. 2004
RNA amplification and labeling of RNA probes
This protocol generates dye-labeled antisense RNA probes for microarray hybridizations. In a first step polyA-RNA is amplified by in vitro transcription (according to Eberwine and collaborators). During in vitro transcription aminoallyl-UTP is incorporated into the newly synthesized RNA. NHS-ester dyes are then directly coupled to the modified bases in a simple chemical reaction.
Protect all dye-containing solutions from light to prevent photo bleaching. We are using total RNA purified on Qiagen RNeasy columns as starting material but other RNA isolation methods may work as well.
1: Mix: Total RNA 3-10 mg
Control RNA (optional) x ml
RNase-free H2O to 12 ml
2: Incubate at 70ÁC for 10 min. Quick-chill on ice. Collect the contents of the tube by quick centrifugation.
3: Add: 5X First Strand Buffer 4 ml
0.1 M DTT 2 ml
dNTP-mix (10 mM each) 1 ml
4: Incubate at 37ÁC for 2 min to equilibrate the temperature.
5: Add 1 ml of Superscript II and mix gently.
Final volume of the 1st strand reaction: 20 ml
6: Incubate at 37ÁC for 1 hour. Put on ice.
7: Second strand synthesis.
Add: H2O 92 ml
5X Second Strand Buffer 30 ml
dNTP-mix (10 mM each) 3 ml
E.coli DNA Polymerase I 4 ml
E.coli RNase H 1 ml
8: Incubate at 16ÁC for 2 hours. Do not let the temperature rise above 16ÁC.
9: Add 10 ml of 0.5 M EDTA to stop the reaction.
1: Transfer reaction mix into a phase-lock gel tube (spin tubes briefly before use to collect the gel on the bottom).
2: Add 160 ml of phenol:chloroform:isoamyl alcohol (25:24:1). Mix thoroughly, but do not vortex. Spin 5 min at 14k rpm (12,000-16,000 x g).
3: Pipet off aqueous layer and place in a fresh tube.
4: Add an equal volume (~ 160 ml) of 5M NH4OAcetate.
5: Add 2.5x volumes of 100% EtOH (~ 800 ml). Add 1 µl of Linear Polyacrylamide. Mix and spin for 5 min at 14k rpm, RT.
6: Remove supernatant carefully and wash pellet with 500 ml of 80% EtOH. Do not vortex.
7: Spin for 5 min at 14k rpm.
8: Repeat the wash step.
9: Dry pellet in a speed-vac.
10: Resuspend pellet in 10 ml of RNase-free H2O.
Use AmbionÍs Megascript T7 kit and aminoallyl-UTP. Set up reaction at room temperature.
1: Mix the following:
RNase-free H2O 2.5 ml
ATP solution (75 mM) 2 ml
GTP solution (75 mM) 2 ml
CTP solution (75 mM) 2 ml
UTP solution (75 mM) 1 ml
aa-UTP solution (50 mM) 1.5 ml
10X reaction buffer 2 ml
cDNA from previous step 5 ml
FINAL VOLUME: 20ml
2: Incubate the reaction at 37ÁC for 6 hours to overnight.
3: Clean up the RNA on a Qiagen RNeasy column:
- Add 80 ml of RNase-free water
- Add 350 ml of buffer RLT to the sample (add b-ME to buffer RLT before use; 10 ml b-ME / 1 ml buffer).
- Add 250 ml 100% EtOH to the sample. Mix well by pipetting.
- Transfer sample (700 ml) to an RNeasy mini spin column.
- Centrifuge for 15 sec at full speed.
- Reload column with flow-through and spin for 15 sec at full speed. This step may increase the RNA yield.
- Transfer RNeasy column to a new 2-ml collection tube (supplied in kit)
- Add 500 ml of phosphate wash buffer. (DonÍt use QiagenÍs buffer RPE since it contains Tris that might interfere with the labeling reaction). Centrifuge for 15 sec at 14 k (>8,000 x g), and discard flow-through
- Pipet 500 ml of phosphate wash buffer onto the column. Centrifuge for 2 min at maximum speed. Discard flow-through.
- Centrifuge at full speed for 1 min.
- Transfer RNeasy column into a 1.5 ml collection tube.
- Add 30 ml of RNase-free water directly onto the RNeasy membrane.
- Centrifuge for 1 min at full speed.
- Repeat elution step with another 30 ml of water, into the same collection tube. Final volume: 50-60 ml.
4: Run 2 ml of the RNA on a 1% agarose gel or run an aliquot on a Bioanalyzer. Determine RNA concentration and calculate total yield. The transcription reaction should result in at least 30-50 mg of RNA. Store RNA at Ü80oC until use.
Important: Make sure, to never over-dry the RNA in the following steps. Precipitated RNA will appear as colored speckles on the membrane of the RNeasy column after elution.
1: Dry down 5-10 mg of RNA in a speed-vac to 3 ml.
2: Add 1 ml of 0.4 M Na2CO3 pH 8.5. Vortex vigorously to resuspend any precipitated RNA.
3: Add 4 ml of dye solution, mix by vortexing and incubate for 1 h in the dark.
4: Add 92 ml of RNase-free water and purify RNA on an RNeasy column as described above. Use buffer RPE for the wash steps. Elute twice with 30 ml of RNase-free water.
Check the membrane for colored speckles (see above).
5: Measure dye incorporation and RNA recovery by spectrophotometry: Dilute 4 ml of the eluate into 46 ml of water and analyze the sample in a spectrophotometer using a micro-cuvette. For Cy3-containing samples measure the absorbance at 260 and 550 nm and for Cy5 at 260 and 650 nm. Calculate the dye incorporation as follows:
dye molecules per 1000 nt= (Adye/A260)‡(9010 cm-1M-1/edye)‡1000
with eCy3= 150,000 cm-1M-1 and eCy5= 250,000 cm-1M-1.
labeling reaction normally incorporates 25-50 dye molecules per 1000 nt.
1: Combine the labeled RNAs of a sample pair (~110 ml). Dry down RNA solution to 9 ml in a speed-vac. To avoid over-drying, take the tubes out of the speed-vac a few time during the drying procedure and vortex them thoroughly.
Add 1 ml of 10X fragmentation buffer (Ambion) and mix by vortexing. Incubate at 70oC for exactly 10 min. Vortex the tubes for a few seconds. Put tubes on ice and add 1 ml of stop buffer.
Note: This method generates RNA fragments <200 nt (peak at ~85 nt).
- Pre-spin a Spin-50 column in a microcentrifuge at 1000 g for 3 min. Empty collection tube.
- Add 500 ml of RNase-free water to the column and spin column in a microcentrifuge at 1000 g for 3 min.
- Discard collection tube and transfer column to an amber tube.
- Load RNA sample onto the center of the column. Spin column in a microcentrifuge at 1000 g for 3 min.
3: Continue with the hybridization protocol for RNA probes.
E.coli Ribonuclease H (Cat. No. 18021071); 120 units, enough for 60 rxns
E.coli DNA Polymerase I (Cat. No.18010025); 1000 units, enough for 25 rxns
Superscript II (Cat. No.18064071); 4x10,000 units, enough for 200 rxns
dNTP set (100 mM) (Cat. No. 10297018)
Megascript T7 kit (Cat. No. 1334), enough for 40 rxns
Fragmentation reagents (Cat. No. 8740), enough for 200 rxns
Aminoallyl-UTP (Cat. No. 8437), enough for 33 rxns
Cy3 Mono-Reactive Dye Pack (Cat. No. PA23001)
Cy5 Mono-Reactive Dye Pack (Cat. No. PA25001)
Phase Lock Tubes, Gel Light (200), 1.5 ml tubes (Cat. No. 0032 007.961)
RNeasy Mini Kit (250) (Cat. No. 74106)
Linear Polyacrylamide (Cat. No. 5-6575)
Spin-50 Mini-Column (Cat. No. 1415-1602)
T7dT primer (PAGE purified):
5Í- TCT AGT CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG GCG TTT TTT TTT TTT TTT TTT TTN N-3Í
Phenol:chloroform:isoamyl alcohol (25:24:1).
5x Second Strand Buffer (store at -20ÁC):
100 mM Tris-HCl pH 6.9
450 mM KCl
23 mM MgCl2
50 mM (NH4)SO4
0.75 mM b-NAD+
0.1 M Na2CO3 pH 8.5:
Prepare fresh buffer every 3-4 weeks. Make up with RNase-free water.
Phosphate Wash Buffer:
Prepare: 1 M K2HPO4 and 1 M KH2PO4 solutions using RNase-free water.
Mix 9.5 ml of 1 M K2HPO4 and 0.5 ml of 1 M KH2PO4 to generate 1 M KPO4 pH 8.5 buffer.
For 100 ml of wash buffer mix:
0.5 ml 1 M KPO4 pH 8.5
80 ml 100% ethanol
19.5 ml RNase-free water
Cy-dye solutions (store at -20ÁC protected from light):
Resuspend the dried dye of one vial in 73 ml of DMSO. Avoid repeated thawing of the dye solutions.