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

Kenneth J Livak,Quin F Wills,Alex J Tipping,Krishnalekha Datta,Rowena Mittal,Andrew J Goldson,Darren W Sexton,Chris C Holmes

doi:10.1016/j.ymeth.2012.10.004

Abstract

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.

Highlights

Single-cell analysis has been called ‘‘the new frontier in Omics’’ [1]
In the study of single-cell gene expression, one of the most provocative findings is that eukaryotic transcription occurs in pulses
As data were collected for 96 assays, there are a total of 138,240 qPCR data points

Summary

Introduction

Single-cell analysis has been called ‘‘the new frontier in Omics’’ [1]. For a variety of reasons and using a variety of techniques, researchers are analyzing cellular heterogeneity by collecting genomics data at single-cell resolution [2]. In the study of single-cell gene expression, one of the most provocative findings is that eukaryotic transcription occurs in pulses. This is shown most directly by the results of Chubb et al [3]. They detected nascent transcripts of dscA, the discoidin Ia gene, directly in living Dictyostelium cells. For this gene, they measured a mean burst duration of 5.2 min and a mean interval of inactivity of 5.8 min, but there was a great deal of stochastic variation in each of these parameters. It is important to note that they were able to detect nuclear transcripts because of the high intensity caused

Methods

Results

Conclusion