Section on Neural Gene Expression

PREPARATION OF RNA PROBES (RIBOPROBES) FOR HYBRIDIZATION HISTOCHEMISTRY

Typically, the plasmid containing the cDNA to be transcribed is linearized with a restriction enzyme. It may be useful to restrict a large amount (about 10µg) of plasmid if that particular probe is to be repeatedly made. Another approach is to generate PCR fragments of the desired cDNA using primers that contain different RNA polymerase transcription initiation sequences. The PCR fragments are gel-purified and used below instead of the linearized plasmid.

Materials

5X transcription buffer (200mM Tris-HCl, ph 7.9, 30mM MgCl₂, 50mM NaCl, and 10mM spermidine)
100mM dithiothreitol (DTT)
10mM ATP
10mM CTP
10mM GTP
alpha-³⁵S-UTP (>1000Ci/mmol)
RNasin (Promega)
DNase (RNase-free)
15 U/µl RNA polymerase (SP6, T3, or T7)
TE (10mM Tris-HCl, 1mM EDTA, pH 7.6)
4M NaCl
25µg/µl yeast tRNA
Ethanol
5M DTT
10% sodium dodecyl sulfate

Preparation of probe
1. Add ³⁵S-UTP and about 250 ng of linearized plasmid (or 10-100 ng of PCR product) to microfuge tube. Dry in vacuum centrifuge at room temperature.

The final concentration of alpha-³⁵S-UTP in the reaction should be between 20 and 30µM. If alpha-³²P-UTP is used to make probes for northern analysis, use 5 µl of the radiochemical and add 0.5 µl of 150µM UTP to the components in step 2 below (15µ M cold UTP and 3µ M ³²P-UTP (500Ci/mmol) final concentrations).

2. Add, at room temperature, to the tube with the dried alpha³⁵S-UTP and DNA: 1 µl 5X transcription buffer, 0.5 µl 100mM DTT, 0.5 µl 10mM ATP, 0.5 µl 10mM CTP, 0.5 µl 10mM GTP, 0.5µl RNasin, 0.5 µl RNA polymerase and H₂O to 5 µl. Mix thoroughly, especially where the ³⁵S-UTP and DNA has pelleted.
3. Incubate at 37° C for 30 min. and add another 0.5µl of the appropriate RNA polymerase.
4. Incubate at 37° C for 30 min. and add 0.5 µl RNasin and 0.5 µl DNase (0.5U). Incubate at 37° C for 10 min.
5. Add 420 µl TE, 25 µl 4M NaCl, 2 µl tRNA and 1 ml ethanol. Mix thoroughly, place on wet ice for 10 min. and spin in a microcentrifuge at 13,000 rpm for 10 min.
6. Remove the supernatant and rinse the pellet with 1 ml of 70% ethanol. Remove the ethanol and dissolve the probe in 485 µl TE, 10 µl 10% SDS and 5 µl 5M DTT. Count 1 µl of radiolabeled probe in a scintillation counter. Freeze at -80° C until used.

Expect about 1 x 10⁶dpm per µl. One can estimate the specific activity based on the the percentage of Us in the transcript.
Note: The reaction can be further miniaturized to a 1µl volume if several probes are labeled at once with the same enzyme. Dry 2-3µl of alpha-³⁵S-UTP per reaction. Make up a cocktail with transcription buffer, ATP, CTP, GTP, RNasin, RNA polymerase, DTT, and water to 0.75µl per reaction. Use this cocktail to redissolve the ³⁵S-UTP, then put 0.75 µl aliquots in 0.5ml screw-top microfuge tubes. Add 50 ng linearized DNA template and a drop of mineral oil. Spin briefly. Incubate for 60 min. at 37° C, then add 0.25 µl each of DNase and RNasin, spin briefly, and incubate another 10 min. Add 400µl TE, 25µl 4M NaCl, and 50µg of tRNA to the tube. Extract once with chloroform to remove oil, and precipitate supernatant with 1ml ethanol on wet ice. Dissolve the pellet in 94µl TE, 5µl 10% SDS, and 1µl 5M DTT.

7. For digoxigenin-labeled probes, use (final) 100µM UTP and 400µ M digoxigenin-UTP (Boehringer Mannheim). Dissolve the precipitated probe in 50µl TE/0.5% SDS. Use 3-10µl/100µl hybridization solution.

The Section on Neural Gene Expression is in the Laboratory of Cellular and Molecular Regulation, Division of Intramural Research Programs is within the National Institute of Mental Health (NIMH) is a part the National Institutes of Health (NIH), and is a component of the U.S. Department of Health and Human Services.