Materials and Reagents

Linearized DNA template for transcription of the RNA of interest
MAXIscript in vitro Transcription Kit (Applied Biosciences, catalog number: AM1308M ) (This kit contain ATP, CTP, GTP, α-P32UTP and DNase I)
RNasin (Promega Corporation, catalog number: N2611 )
RNase T1 (Applied Biosciences, catalog number: 10109193001 )
0.5 M EDTA (Life Technologies, Gibco^®, catalog number: 15575-038 )
ddH₂O (GeneMate Loyalty, catalog number: UPW-1000 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
MgCl₂ (Sigma-Aldrich, catalog number: M-2393 )
Glycerol (Sigma-Aldrich, catalog number: G7893-1L )
DTT (Sigma-Aldrich, catalog number: D9779-5G )
Tris-Base (Thermo Fisher Scientific, catalog number: BP152-1 )
SDS (Sigma-Aldrich, catalog number: L-4390 )
Bromphenol blue (Sigma-Aldrich, catalog number: B5525 )
TEMED
Ammoniampersulfate
5x binding buffer (see Recipes)
2x SDS loading buffer (see Recipes)
10x TGE buffer (see Recipes)
6% TGE gel (see Recipes)

Equipment

SPIN-PureTM column (G-50) (PireBiotech SCW50-50 DEPC-water)
SDS-PAGE system (Bio-Rad, catalog number: 165-1802 )
UV Stratalinker 1800 (Stratagene, catalog number: 474645 )
Film processor (Konica Minolta, model: SRX-101A )
Centrifuges (Eppendorf, catalog number: 5810R )
Scintillation Counter (Beckman, catalog number: LS6500 )
Bench top Radiation Shield with 6-1/4" base (Cole-Parmer, catalog number: WU-36218-00 )
37 °C water bath

Procedure

In vitro transcription of ³²P-labled RNA probes
1. DNA templates for synthesis of the RNA probe are generated by PCR using specific primers.
2. Thaw frozen reagents, place RNA polymerase on ice and vortex the 10x Transcription buffer and keep the buffer at room temperature. All reagents should be centrifuged briefly before opening.
3. Assemble transcription reaction at room temperature as the following sequence:
  1 μg DNA template
  ddH₂O to 20 μl
  2 μl 10x transcription buffer (after addition of water and DNA)
  1 μl 10 mM ATP
  1 μl 10 mM CTP
  1 μl 10 mM GTP
  5 μl [α-³²P]UTP
  2 μl T7 enzyme mix
  2 μl RNasin (40 U/μl)
  Mix thoroughly.
4. Incubate 1 h at 37 °C.
5. Add 1 μl of 5 mg/ml DNase I to digest the rest template DNA and incubate 10 min in 37 °C water bath.
6. Add 1 μl of 0.5 M EDTA, mix and add 78 μl ddH₂O to adjust final volume to 100 μl and mix.
Purification of ³²P-labled RNA probes with SPIN-Pure column (G-50)
1. Allow a minimum of 30 min of room temperature to warm the columns before the following steps.
2. Gently invert the column several times to suspend the column buffer (DEPC-Water).
3. Remove the top cap from the column, and then remove the bottom tip.
4. Allow the column buffer to drain by gravity before proceeding.
5. Place this column/tube apparatus into an adaptor tube. Centrifuge at 1,100 x g for 2 min at room temperature.
6. Repeat centrifugation again to let the column dry completely and discard the collection tube and the eluted buffer.
7. Put the column in a second collection tube in upright position and apply the RNA sample (20 to 50 μl) to the center of the column gel very slowly and carefully (the rest RNA sample can be stored at -80 °C for one week).
8. Centrifuge at 1,100 x g for 4 min. The purified ³²P-labled RNA probe is collected in the bottom of the collection tube. Discard the spin column and continue with the following procedure.
9. Pick 1 μl of the RNA sample and count the cpm value.
RNA and protein binding reaction
1. Prepare the following mixture on ice:
  ³²P-labled RNA probe (2 x 10⁵ cpm)
  1 μl 12.5 mM ATP
  3μl 5x binding buffer
  3 μl 0.5 M KCl
  Protein (100 ng) (this protein can be a GST fusion protein purified according to the protocol prepared by GE Healthcare Life Sciences)
  H₂O to final volume of 15 μl.
2. Mix gently by pipetting up and down for 10 times, avoiding vortex.
3. Place the uncovered tube containing the reaction mixture on ice, directly underneath the bulb (about 10 cm from the surface) of a 254-nm UV light source, irradiation with 4 x 10⁵ μJ/cm² energy.
4. Incubate at room temperature for 30 min then keep on ice for another 3 h.
Remove the non-incorporated ³²P
1. Add 1 μl of RNase T1 (1 U/μl) to each tube and incubate for additional 10 min at 37 °C to degrade the free RNA.
2. Add an equal volume (16 μl) of 2x SDS loading dye andseparate the sample in TGE-gel or SDS-PAGE (no boiling need).
3. Autoradiography for 12 h to 7 days for visualization.

Recipes

5x binding buffer
50 mM HEPES (pH 7.2)
15 mM MgCl₂
25% glycerol
5 mM DTT
Stored at -20 °C
2x SDS loading buffer
125 mM Tris-Cl (pH 7.6)
20% (v/v) glycerol
4% (w/v) SDS
0.008% (w/v) bromphenol blue
20 mM DTT
10x TGE buffer
Tris 100 mM
Glycine 1 M
EDTA 10 mM
pH 8.3
6% TGE gel
6 ml 30% acrylamide
5 ml 10x TGE buffer
35 ml H₂O
300 μl ammoniampersulfate
22 μl TEMED
Notes:
1. Avoiding RNase contamination is the key for successful.
2. As ³²P is radioactive, this experiment should be performed under the protection of Benchtop Radiation Shield.

Acknowledgments

This work was supported by the National Institutes of Health (R01 CA123490 and R01CA143107 to MZ) and CURE (MZ and LG).

References

Gu, L., Zhang, H., He, J., Li, J., Huang, M. and Zhou, M. (2012). MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells. Oncogene 31(11): 1342-1353.
Walker, J., de Melo Neto, O. and Standart, N. (1998). Gel retardation and UV-crosslinking assays to detect specific RNA-protein interactions in the 5' or 3' UTRs of translationally regulated mRNAs. Methods Mol Biol 77: 365-378.