RNA extracts were dried in 1.5 ml Eppendorf tubes and resuspended in 30 µl of 2 × TESS (10 mM N-Tris[hydroxy-methyl]methyl-2-aminoethane sulfonic acid, pH 7.4; 10 mM ethylenediaminetetraacetic acid [EDTA]; 0.3 M NaCl; 0.5% sodium dodecyl sulfate [SDS]) that contained 150 to 300 K cpm of a probe. Samples were covered with mineral oil and hybridized overnight at 75°C. For RNase treatment, 250 µl of a buffer containing 0.3 M NaCl; 5 mM EDTA; 10 mM Tris-HCl (pH 7.5), 40 µg/ml RNase A (Worthington, Biochemicals, Freehold, NJ) and 2 µg/ml RNase T1 (Calbiochem, San Diego, CA) was added and each sample was incubated at 30°C for 1 hour. Trichloroacetic acid precipitation was effected by the addition of 1 ml of a solution that contained 5% TCA and 0.75% sodium pyrophosphate. Precipitates were collected onto a filter in sets of 24 using a cell harvester (Brandel, Gaithersburg, MD) and were measured in a scintillation counter with liquid scintillant (Beckman, Palo Alto, CA).
