This method is obtained from Bill Clouston's thesis and is essentially that described by Kalinyak and Perlman (JBC 262:460-4, 1987) with the addition of preservative-free sodium heparin to reduce non-specific hybridization.

Method

1. Run Northern in the usual way, transfer to Genescreen Plus in 20x SSC and bake in oven at 80ºC for 2 hours.
2. Hybridization buffer is as follows:

-	50% Formamide
-	3x SSC
-	10x Denhardts'
-	10 mM phosphate buffer, pH 8.0
-	2 mM EDTA
-	0.1% SDS
-	200 ug/ml herring sperm DNA
-	800 U/ml preservative-free sodium heparin (DBL, 25,000U/5ml, Royal Melbourne Hospital Pharmacy)

3. Pre-hyb membrane for 4-6 hours at 60ºC.
4. Hybridize in fresh buffer for 18-24 hours at 65ºC - 20 ng of riboprobe to the bag in a small volume of buffer (eg. 3-5 ml). Riboprobes should prepared exactly as described for hybridization histochemistry. This can include hydrolysis, but this may or may not make a difference to the degree of background.
5. Wash at high stringency, ie. 0.1X SSC, 0.1% SDS at 65ºC. Be careful to wash all formamide-containing hybridization buffer off at low stringency first, ie. use a 2x SSC wash initially then increase stringency progressively. I obtained a good signal with little background using this washing regimen but lost the signal when I washed at 75ºC.
6. Expose against X-ray film using an intensifying screen at -70ºC for 24 hours or longer. I obtained a good signal after 36 hours, equal to that obtained with Northerns using cDNA probes after 96 hours exposure.