α32p labeled dctp

For northern blot analysis, total RNA was extracted from DROSHA KO cell lines 48 hr after transfection with lentiviral packaging plasmids. For the detection of viral genomic RNA in viral particles, virus supernatants were treated with Benzonase (Sigma-Aldrich, Saint Louis, MO, USA) to remove free RNA and DNA, followed by centrifugation at 13,000 × g at 4°C overnight. Total RNA was extracted from viral pellets by concentrating the Benzonase-treated virus supernatant in a Beckmann XL-90 centrifuge using SW-28 swinging buckets. The total RNA was isolated using TRIzol reagent (Ambion, Austin, TX, USA), then resolved on a 15% polyacrylamide Tris-borate-EDTA (TBE) urea gel or formaldehyde/agarose gel for separation of small RNAs or viral genomic RNA, respectively. RNA was transferred to Hybond-N+ nylon membrane (Amersham, Piscataway, NJ, USA) and UV-crosslinked. The blots were prehybridized using PerfectHyb Plus Hybridization buffer (Sigma, St. Louis, MO, USA) at 42°C for 1 hr. The blot was probe-labeled with either γ-32P-labeled ATP (Perkin Elmer) and hybridized at 42°C overnight, or with α-32P-labeled dCTP (Perkin Elmer) at 58°C overnight. Blots were washed in 2× sodium citrate, 0.1% SDS at room temperature, and exposed to film. Forward and reverse sequences for REV-responsive element (RRE) probe were as follows: 5′-GCTTTGTTCCTTGGGTTCTTG-3′ and 5′-CCAGGAGCTGTTGATCCTTTAG-3′.