snoMEN vector targeted to miR21 primary transcript.

<p>(<b>a</b>) Structure for targeted endogenous miR21 primary transcript (mCherry–pri-miR21 snoMEN) and schematic diagram of miR21 maturation pathway. This construct has three snoMEN RNAs (blue pentagons) as previously described[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138668#pone.0138668.ref001" target="_blank">1</a>], except that here the M box sequences are complementary to specific sequences within the endogenous miR21 primary transcript. (<b>b</b>) Validation of snoMEN expression by Fluorescence In Situ Hybridisation (FISH) analysis. Each snoMEN RNA was detected by using a M box specific RNA probe labelled with Cy3 (Cy3). DNA is stained by DAPI (DAPI). Scale bar is 10 μm. (<b>c</b>) RNA analysis. Total RNA from HeLa cells was harvested 24 hours after transfection and qRT-PCR was performed to identify miR21 precursor molecules using miR21 precursor specific primers (pre-miR21). Following cDNA synthesis, qPCR was performed using matured miR21 specific primer and universal primer provided by PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138668#sec008" target="_blank">methods</a>) (Matured-miR21). U3 was used as a loading control. Graph depicts mean and standard deviation from a minimum of 5 independent experiments. (<b>d</b>) HeLa cells transfected with pri-miR21-snoMEN/Control for 24 hours prior to total RNA extraction and northern blot analysis.