Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Summary

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Abstract

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

Introduction

Individual cells in a population can show widely differing responses to a uniform physiological stimulus^1,2,3,4. The genetic variation of cells in a population is one mechanism for this variety of responses, but there are also several non-genetic factors that can increase the variability of responses, even in a clonal population of cells. For example, the levels of individual proteins and other important signaling molecules can vary on a cell-by-cell basis, giving rise to variation in downstream gene expression profiles. Additionally, gene a....

Protocol

1. Advance Preparation

1. Select up to 96 genes of interest whose expression will be measured.
   NOTE: At least one of these genes should be a "housekeeping gene," such as ACTB or GAPDH, that is known to be expressed at a relatively high and constant level under the conditions used in the experiment. This gene will be used to identify positively sorted wells (step 8.1) and amplified samples (step 10.1).
   NOTE: For the example experiment, well-characterized, direct targets of p53 with a variety of known functions^11,14 and GAPDH as a housekeeping control were selected. Please see Reference 11 for....

Results

A general overview of the protocol is shown in Figure 1, including steps for cell treatment, the isolation of single cells by FACS, the generation and pre-amplification of cDNA libraries from single-cell lysates, the confirmation of single-cell cDNA libraries in sorted wells, and the measurement of gene expression by qPCR.

In preparation for single-cell isolation and gene expression analysis, it is necessary to .......

Discussion

We have presented a method for isolating individual mammalian cells from a population of adherent cells grown in culture and for assaying the expression of approximately 96 genes in each cell. Good advance preparation is critical for this method to work well. In particular, designing and testing primer pairs specific to the transcripts of interest (steps 1.2-1.3) are time-consuming but important steps, as the primers determine the quality of the single-cell measurements. Once reliable primer pairs have been obtained, the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNeasy Plus Mini Kit	Qiagen	74134
High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor	ThermoFisher	4374966
Phusion High-Fidelity DNA Polymerase	New England BioLabs	M0530S
QIAquick Gel Extraction Kit	Qiagen	28704
Quant-iT High-Sensitivity dsDNA Assay Kit	ThermoFisher	Q33120
2.0 ml low adhesion microcentrifuge tubes	USA Scientific	1420-2600
DNA Suspension Buffer	Teknova	T0221
Axygen 0.2 ml Maxymum Recovery Thin Wall PCR Tubes	Corning	PCR-02-L-C
GE 96.96 Dynamic Array DNA Binding Dye Sample & Assay Loading Reagent Kit	Fluidigm	100-3415
HyClone RPMI 1640 media	GE Healthcare Life Sciences	SH30027.01
Fetal Bovine Serum, Certified (US)	ThermoFisher	16000-044
Antibiotic-Antimycotic Solution	Corning	30-004-CI
Neocarzinostatin	Sigma	N9162
ELIMINase	Decon Labs	1101
SUPERase-In	ThermoFisher	AM2696
CellsDirect One-Step qRT-PCR Kit	ThermoFisher	11753500
E. coli DNA	Affymetrix	14380 10 MG
ThermalSeal Sealing Film, Sterile	Excel Scientific	STR-THER-PLT
BD FACSAria IIu	BD Biosciences
HyClone Trypsin 0.05%	GE Healthcare Life Sciences	SH30236.01
PBS, 1x	Corning	21-040-CV
Falcon 40 µm Cell Strainer	Corning	352340
Exonuclease I	New England BioLabs	M0293S
SsoFast EvaGreen Supermix with Low ROX	Bio-Rad	172-5210
96.96 Dynamic Array IFC for Gene Expression (microfluidic qPCR chip)	Fluidigm	BMK-M-96.96
IFC Controller HX (loading machine)	Fluidigm
BioMark or BioMark HD (microfluidic qPCR machine)	Fluidigm
Real-Time PCR Analysis software	Fluidigm
MATLAB software	MathWorks