hMSC differentiation marker detection using Thermo Scientific SolarisTM qPCR Gene Expression Assays

Although microarrays are useful for rapid whole-genome profiling, a complementary method with improved sample throughput, sensitivity and dynamic range is needed for follow-up studies. Quantitative real-time PCR (qPCR) is often the method of choice to validate gene expression results from whole-genome microarrays. SolarisTM qPCR Gene Expression Assays are predesigned on a genome-wide scale using a novel, tier-based algorithm to detect all variants of a given gene and distinguish among closely related family members. Solaris assays incorporate minor groove binder (MGBTM)1 and SuperbaseTM technologies (Epoch Biosciences, Inc) for increased sequence design space and enhanced specificity. Combining these two chemical strategies with a fluorescent (FAM) reporter dye and corresponding Dark QuencherTM fluorochrome (Epoch Biosciences, Inc) results in highly specific and sensitive assays that consistently function under universal thermocycling conditions. Here we describe an application of Solaris technology to validate the microarray expression data from early-stage osteogenic human mesenchymal stem cells (hMSCs). microRNAs (miRNAs) are involved in many aspects of cellular processes; however, little is known about their role in the regulation of adult stem cell differentiation. In a recently published screen using a Thermo Scientific Dharmacon miRIDIAN microRNA Inhibitor and Mimic library, miR-148b was shown to increase alkaline phosphatase (ALPL) activity, an early marker of osteoblast differentiation2. Here we show how the novel Solaris platform was used to further characterize gene expression in hMSCs treated with differentiation medium or with miRNA mimics. We assessed osteogenic differentiation in hMSCs treated with medium or with miRNA mimics (Fig. 1). For the medium treatments, hMSCs were grown in osteoblast differentiation medium or propagation medium. For the mimic treatment, we transfected miRIDIAN miR-148b mimic or miRNA mimic negative control 1 into hMSCs. Six days after induction of osteogenic differentiation, we collected the cells and assessed the hMSC differentiation marker detection using Thermo Scientific SolarisTM qPCR Gene Expression Assays