We have used a novel real time PCR approach to profile global changes in microRNA expression during adipocytic differentiation of 3T3-L1 fibroblast cells. This profiling strategy makes use of a novel two stage screen resulting in a substantial increase in throughput and decrease in reagent use. In the first step, cDNA is prepared from individual samples of undifferentiated fibroblast, and cells at early and late stages of Adipocyte differentiation. A pooled cDNA sample prepared by pooling equimolar amounts of individual cDNAs was used in an initial profiling experiment to identify microRNAs that are expressed at detectable levels in the experimental system. Profiling was performed in triplicate with real time PCR assays for 591 mouse miRNAs (miRBase v12.0). In the second stage of screening, individual cDNA samples were each analyzed in triplicate using the assays for miRNAs that showed expression in the pooled cDNA sample. Expression data was normalized against a panel of small non-coding RNAs and was expressed as x-fold changes relative to expresion in undifferentiated 3T3L1 cells. Our results indicate that 192 (∼32%) miRNAs described in miRBase v12.0 were detectable in resting and differentiated 3T3-L1 cells, and of these, 38 (or 20%) showed differential regulation during adipogenesis (x-fold change>3.0). When compared to data from previous array-based profiling studies, our screen identified additional differentially regulated miRNAs. In addition to profiling changes in mature miRNA levels, miRNA precursor stem loop transcripts were also quantified to gain insight into post transcriptional regulation (if any) of miRNA biogenesis during adipocyte differentiation. The high specificity and sensitivity of the miScript RT-PCR system, together with a novel sample-pooling strategy makes this system a method of choice for miRNA expression profiling. Pooled primary profiling can substantially reduce the total number of PCR reactions which need to be performed and increases assay throughput.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3033.