Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells

► Microfluidic arrays enable analysis of 96 qPCR assays on 1440 single cells. ► Detailed methods on obtaining qPCR data and performing preliminary data processing. ► Data from sufficient cells to address noise inherent in single-cell transcription. ► Methods used for conventional qPCR do not necessarily apply to single-cell qPCR. The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR.