In vitro primary and precursor miRNA processing assays

The DNA segments containing pri-miRNA sequences of miR-20b and miR-107 were amplified from genomic DNA by PCR, then cloned into a pUC19 vector downstream of a T7 promoter. The plasmids were linearized and gel purified. The internally labeled pri-miRNAs were transcribed in vitro using T7 RNA polymerase (Promega) in the presence of [α-³²P]-CTP, and purified with QIAGEN RNeasy Mini kit. HeLa cell nuclear extracts from ProteinOne were used for the pri-miRNA processing assays. Processing assays were carried out (typically using 25 μg cell nuclear extract per 20 μl reaction) at 30°C for 1 h. Samples were then phenol/chloroform extracted, precipitated and re-suspended in RNA loading dye prior to denaturing polyacrylamide gelelectrophoresis separation. All the assays were preformed at least in triplicate.

Processed pri-miRNA products were resolved on 12.5% acrylamide-8 M urea gels together with Molecular weight markers of radiolabeled RNA oligonucleotides (Ambion® Decade™ Marker System). After electrophoresis, the gels containing the separated RNAs were exposed to a phosphor-screen and visualized by autoradiography using a Storm PhosphorImager (GE Life Sciences). Band intensities were quantified using ImageJ software (31).