6514 Nucleic Acids Research, 2008, Vol. 36, No. 20 Figure 1. Processing and eﬃcacy of microRNAs in vivo. (A) Northern blot analyses of miRNA expression in wild-type and mHelicase cells. For miR- 21, small RNAs were harvested from wild-type and Dicer mHelicase cells. The unprocessed pre-miR-21 (arrowhead) and mature miR-21 species (arrow) are indicated. The asterisk denotes a nonspeciﬁc signal that runs slightly >70 nt, which likely represents cross hybridization of the 20-O- methyl-modiﬁed oligonucleotide to an abundant tRNA, serves as an internal loading control. The illustrated size marker is based on the migration of the Ambion Decade ladder. For miR-23a and miR-27a, total RNA was harvested from wild-type and mHelicase cells transfected with either the control U6 vector (Control) or vectors expressing U6-driven shRNA (SII-c21) or lhRNA (SII-c50). Arrows denote the migration of the mature miR- 23a and miR-27a. For miR-27a, the 63 nt pre-miR-27a is indicated by the arrowhead. The illustrated size marker is based on the migration of the Ambion Decade ladder. U6 snRNA expression was also detected as a loading standard. (B) Inhibition of miRNA gene expression by an ectopic miRNA expression construct. Twenty-ﬁve nanograms of the psiCHECK-miR-93 reporter vector and 100 ng of the MCM7 miRNA expression vector were co-transfected with an empty vector or a vector expressing the wild-type Dicer cDNA into wild-type or Dicer mHelicase cells. Luciferase values were determined 24 h later and the Renilla target was normalized to Fireﬂy luciferase, which is contained on the same reporter construct but is not subject to downregulation by the miRNA. Luciferase values in response to the irrelevant HIV miRNA expression vector (HIV) were set at 100%. The mean and standard deviations from three experiments are presented. P < 0.02, P < 0.05. labeled pre-miRNA was incubated with 1 ml of rDicer (Ambion) according the manufacturer’s instructions. For in vitro reactions using cell extracts from wild-type or Dicer mHelicase cells, 5 104—1 105 c.p.m. of labeled pre-miRNA was incubated with 25 mg of cell extract, 3.2 mM MgCl2 and 0.5 mM ATP at 378C for 1–3 h. Reactions were stopped by the addition of EDTA and immediately run on 10–15%/8 M Urea denaturing polyacrylamide gels. Following electrophoresis, separated RNAs were analyzed by exposing the gel to a phosphorscreen and analyzed using a Storm phosphorimager. The processing eﬃciency was determined as follows: processing eﬃciency = matureIDV/(matureIDV + precursorIDV) IDV, Integrated Density Value. For in vitro analysis of sh- and lhRNAs, PCR products that include the T7 phage promoter were generated for SII-rev 21-, 50- and 80-bp hairpin sequences. The primers for producing the T7 transcription units were: upper (50-TAATACGACTCACTATAGCCTGTGTCTCTTCA GTTACT-30) and lower (50-AAGCCTGTGCCTCTTC AGCTACC). Individually sized sh- or lhRNA RNAs were transcribed using the MAXIscript T7 kit (Ambion)