Objective

Quantitative PCR has been regarded as the gold-standard for assessing messenger RNA levels in cultured cells and tissues. However, the technique has several disadvantages. The reverse-transcriptase and amplification steps can bias transcript quantitation, and the limit on the number of transcripts which can be multiplexed results in a combination of relatively low throughput with large RNA input, impacting on cost and use of precious samples. In order to overcome these limitations, we assessed the suitability of a branched DNA assay alongside qPCR as a method to quality control the differentiation of stem cells into neurons using a published small molecule differentiation protocol. A panel of 43 genes linked to markers of differentiation was tested in parallel with both techniques. Results from both methods were comparable - the bDNA assay producing consistent data but using significantly less sample (1-2 wells from a 96-well plate for all 43 markers vs. 48 wells in qPCR). The higher throughput and lower sample requirements for the bDNA protocol allows its use as a continual QC tool rather than as a retrospective analysis of differentiation resulting in more efficient, accurate monitoring of neuronal differentiations and a subsequent reduction of wasted experimental time on substandard neuronal populations.