NORTHERN ANALYSES USING OLIGODEOXYNUCLEOTIDE PROBES AND RIBOPROBES

Northern analysis of RNA samples using the same probe sequences is a useful control. Although DNA/RNA or RNA/RNA hybridizations on synthetic membranes are unlikely to kinetically identical to those performed on tissue sections, they are likely to be similar under the same temperature, salt, and formamide conditions. Furthermore, if bands representing mRNA sizes different from what have been reported previously (probably using double-stranded cDNA probes) are observed, then one must consider the likelihood that the hybridization histochemistry is also detecting these other transcripts which may or may not be transcribed from the same gene.

Northern analyses using oligodeoxynucleotide probes
Materials

GeneScreen® (New England Nuclear)
Northern hybridization solution (see recipe)
20x SSPE
10% SDS

1. Transfer size-fractionated RNA to GeneScreen® membrane (see Unit )
2. UV cross-link and vacuum-bake filter at 70° C for 2 hrs.
3. Add about 0.5-1ml northern hybridization buffer per 10 cm2 of membrane and incubate 3 hrs. at 37° C.

The prehybridizations, hybridizations and washes may be performed conveniently in small roller bottles in an oven.

4. Remove buffer and replace with about 2x106 dpm of probe per ml northern hybridization buffer (use 0.5ml per 10 cm2 of membrane) and incubate at 37° C for 18 hrs.

5. Rinse twice briefly with 1x SSPE/0.2% SDS at 55° C. and then wash 5 times with 15-min. changes of 1x SSPE/0.2% SDS at 55° C.
6. Place the membrane between plastic wrap in a cassette with intensifying screens and appose to film at -80° C.

Northern analyses using riboprobes
Materials

GeneScreen® (New England Nuclear)
Northern hybridization solution (see recipe)
20x SSPE
10% SDS

1. Transfer size-fractionated RNA to GeneScreen® membrane (see Unit )
2. UV cross-link and vacuum-bake filter at 70° C for 2 hrs.
3. Add about 0.5-1 ml northern hybridization buffer per 10 cm2 of membrane and incubate 18 hrs. at 65° C.

The prehybridizations, hybridizations and washes may be performed conveniently in small roller bottles in an oven. If performed in plastic bags, double-bag the membranes.

4. Remove buffer and replace with about 2x106 dpm of probe per ml northern hybridization buffer (use 0.5ml per 10 cm2 of membrane) and incubate at 65° C for 24 hrs.

5. Rinse twice briefly with 0.1x SSPE/0.2% SDS at 65° C and then wash 4 times with 30-min. changes of 0.1x SSPE/0.2% SDS at 65° C.

Never let the membranes cool below 65 degrees (this is done by placing the plastic bags in 65° C water in a large beaker, cutting open the ends of the bags above the waters surface, and pulling the membranes out and immediately placing them in the 65° C wash buffer. This is quite easy with roller bottles.)

6. Place the membrane between plastic wrap in a cassette with intensifying screens and appose to film (e.g., Kodak Biomax MR) at -80° C.

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