Method

Transcribing the probe

1. Linearize the vector with restriction enzyme
   template DNA 18 µl ( 12 µg)
   spermidine (33mM = 10x) 2 µl
   10x reaction buffer 2 µl
   restriction enzyme 2 + 1 µl
   Incubate 1-3 h 37°C. After 1 h add the additional enzyme.
   After 2h test 1µl on a microgel; the template should be 
   completely linearized.
   If you want to keep the template for several weeks you can do phenol 
   extraction and ethanol pricipitation of the linerarized template. 
   I found it unnecessary.
   1µg template is enough for 20 tissue RNA hybridizations.
   
   
2. Make hot probe
   4 µl 5x transcription buffer (BRL)
   2 µl DTT (0.1M)
   4 µl ATP, UTP, GTP (3.3 mM each) (Promega)
   2.4µl CTP (100 µM) (Promega)
   1 µl RNasin (40 U/µl) (Promega)
   5 µl 32P CTP (800 Ci/mmol) (Amersham)
   2 µl template (=1µg)
   add last to avoid pricipitation with spermidine in transcription buffer.
   1 µl RNA polymerase: either T7 (NEB,20.000 U/ml or SP6 (BRL, 15.000 U/ml)
   Incubate 1h 37°. Determine cpm incorporated. Expect 1-2 x10⁶ cpm in 1 µl.
   
   Digest DNA with RQ1 DNase (1U/µl, Promega) and incubate 10 min. 37°
   I added between 1 and 10 µl. It is important that all DNA is digested.
   Remaining template causes nonspecific hybridization.
   
   Phenol extract and ethanol-precipitate the hot probe. I extract twice with
   phenol-Sevag and once with Sevag. Then I dilute the probe (20µl + 300µl DEP H2O),
   add 33 µl 3M acetate, pH 6 and 750 µl ethanol.
   Note, the probe does not last longer than 1 or 2 days because of radiolysis.
   If you need to store the probe longer (2-5 days) do not precipitate but keep the
   probe in 30 % ethanol then there is less radiolysis. Make ppt. when you need the probe.

Use total tissue- or cellular RNA that is in 75% EtOH (i.e. precipitate.). Mix the equivalent of 2 - 20 µg (~20µl) with the hot probe of 1 - 2 x 10⁶ cpm (50µl). Add tRNA as carrier, 10 µg, if the tissue RNA is less than 10 µg. Spin down the combined EtOH precipitate. (cold). Remove supernatant and dry pellet (2 min.vacuum). Add to pellet 30 µl premixed buffer + formamide (20% 5 x hyb buffer, 80% formamide) and vortex very thoroughly 30 - 60 sec.
(Use good formamide (Boehringer) and store aliquots of 1ml in DEP water-treated and baked glass vials.Store at -20°.)

5x hyb.-buffer: 200mM Pipes
2 M NaCl
5mM EDTA 
pH 6.7 Store at RT.
Stocks: 1 M Pipes pH 6.6 in DEP-water and Millipore filter
2.5 M NaCl: DEP-treat and autoclave
0.5 M EDTA pH 8.0, made from autoclaved stock with DEP-water. Adjust pH with conc. HCl
Denature RNA at 85 - 90^o. Do not boil. Then quickly transfer to 50^o water bath. 
Do not let cool below 50^o. 
Incubate ON or about 18h.

Digestion of single strand RNA

Bring samples to RT or put on ice. Add 300 µl ice cold digestion buffer with RNases: 4 µg RNase A and 1000 U RNase T1/ml. 

Digestion buffer: 10mM tris-HCl pH 7.5 (1 ml 1M stock)
5mM EDTA pH 8.2 (1 ml 0.5 M stock)
0.2 M NaCl (4 ml 5 M stock) 
0.1 M LiCl (10 ml 1 M stock)
(water to 100 ml)

Premix: 6 ml digestion buffer
3 µl RNase T1 (BRL or any 2000 U/µl)
24 µl RNase A (Boehringer):10mg/ml boiled (if necessary)

PAGE analysis of RNase  protection fragments

Incubate for 1 hr at RT (some use 37°).
Add 5µl proteinase K, 10mg/ml and 10 µl of 20% SDS (or 15 µl both premixed)
Think what controls you may need.
Hot probe, no RNA, no RNAses to see size of full length transcript.
Hot probe, no RNA but + RNases to see whether digestion works. This should have no bands at all.

Immediately add tRNA, 2 µl 10mg/ml and do phenol-extraction as before: 2 x phenol-Sevag and 1x Sevag. EtOH. ppt. - spin - dry (2min vacc.).  Add 6 µl loading buffer (80-90 % formamide, 10 - 20 % dye); mix - boil 2 min. - spin - load on Urea gel. (5 or 6 % acrylamide, 
8 M urea. For one 0.75 mm gel, 6 ml: 3g urea (sequencing grade)
0.6 ml 10 x TBE
water to 5.2 ml
dissolve at 68° and cool to RT
0.75 ml 40% acrylamide
60 µl 10% APS
6 µl TEMED
When gelled, rinse with water while comb is still in to remove urea.

Before loading, rinse wells thoroughly with running buffer (TBE)
Run at 50-100 V for 1 - 2 hr with TBE. Fix, dry, put on film