Frank Wellmer

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.

 

METHOD:

 

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.

 

I: cDNA synthesis

 

1: Mix:                  Total RNA                                            3-10 mg

                                    T7dT primer (0.5mg/ml)               1 ml

                                    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

 

                                    Final volume of the 2nd strand reaction: 150 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.

 

II: Purification of cDNA

 

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.

 

 

III: In vitro transcription

 

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

T7 Enzyme                                           2 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.

 

IV. RNA labeling

 

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.

 

The labeling reaction normally incorporates 25-50 dye molecules per 1000 nt.


V. Probe hydrolysis

 

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).

 

2: Add 20 ml of RNase-free water to the sample.

-           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.

 

 

MATERIALS AND REAGENTS:

 

Invitrogen:

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)

 

Ambion:

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

 

Amersham:

Cy3 Mono-Reactive Dye Pack (Cat. No. PA23001)

Cy5 Mono-Reactive Dye Pack (Cat. No. PA25001)

 

Eppendorf:

Phase Lock Tubes, Gel Light (200), 1.5 ml tubes (Cat. No. 0032 007.961)

 

Qiagen: 

RNeasy Mini Kit (250) (Cat. No. 74106)

 

Sigma:

Linear Polyacrylamide (Cat. No. 5-6575)

 

USA Scientific:

Spin-50 Mini-Column (Cat. No. 1415-1602)

 

Various sources:

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).

 

SOLUTIONS:

 

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.