The basic Northern blotting technique is that described
in (Ref. 2). Good results can be obtained by analysing between 10 µg and 30 µg of total RNA on a formaldehyde
gel. Denaturing gels are prepared using 1% agarose (molecular biology grade), 7.2% (v/v) formaldehyde (stock solution is 40%) in 1.1 × MOPS buffer pH 7.0. The RNA is
dissolved in 50% deionised formamide (Sigma), 7.2% (v/v)
formaldehyde and 0.5 × MOPS buffer and heated to 65◦C
for 15 minutes. To each sample is added 2 µl of sample loading buffer and 1 µl of ethidium bromide (1 mg/ml), and
the sample is loaded immediately and electrophoresed at
20 V/cm in 1 × MOPS buffer, until the fast blue (bromophenol blue) is about 3 cm from the bottom of the gel. The
RNA is transferred to a membrane by conventional capillary
blotting (Ref. 2), as shown in (Fig. 1). The membrane to be
used for transfer is a matter of personal preference, however
DuralonTM (Stratagene) gives consistently good results. The
manufacturers recommendations for transfer and hybridisation conditions should be followed for optimal results.