Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR)

doi:10.1016/S1383-5726(98)00009-0

Mutation Research/Mutation Research Genomics

Volume 406, Issues 2–4, August 1999, Pages 45-54

https://doi.org/10.1016/S1383-5726(98)00009-0 Get rights and content

Abstract

The determination of the gene expression pattern of single cells has important implications for many areas of cellular and developmental biology including lineage determination, identification of primitive stem cells and temporal gene expression patterns induced by changes in the cellular microenvironment. Global Single Cell Reverse Transcription-Polymerase Chain Reaction (GSC RT-PCR) enables the study of single cell gene expression patterns. Initial observations of significant heterogeneity among single cells derived from a population of cells prompted us to determine how much of this observed heterogeneity was due to the intrinsic variation within the method. In this paper we discuss the sensitivity of GSC RT-PCR for analysis of differences in gene expression between single cells and, in particular, detail the amount of variation generated by the method itself. We found that most of the intrinsic variation in the method occurred in the PCR step. The total variation induced by the method was in the range of 5 fold. While we have determined that there is a five fold methodological variation in GSC RT-PCR, any method which use its components (including generation of cDNAs for microarray analysis) is likely to be affected by such experimental variability, which could limit the interpretation of the resulting data.

Introduction

Many aspects of both normal development and disease result from changes in the expression of multiple genes in individual cells [1]. It is widely accepted that cancer is a clonal disease (i.e., it arises from a single cell) and that tumor progression occurs as a result of molecular changes in individual cells [2]which confer a growth advantage. However, until recently most methods which attempted to study these molecular changes by necessity had to examine populations of cells. An obvious problem with using populations of cells for such studies is that only changes which have occurred in the majority of cells are likely to be identified and differences between individual cells will be difficult or impossible to detect.

To overcome the difficulties with `bulk' methods, techniques have been developed to study gene expression at the single cell level. Disease processes in which multiple changes in gene expression have taken place (for example, colon cancer progression [3]) have been studied using in situ hybridization [4]and in-situ PCR [5]. Very few of these studies have demonstrated strict quantification. Furthermore, with the proliferation of new methods of measuring gene expression (i.e., cDNA microarrays¹ [6]and SAGE 7, 8) the issue of determining real variability in gene expression versus experimental variability has become increasingly important.

One method which allows the examination of relative differences in gene expression between cells is global single cell reverse transcription-polymerase chain reaction (GSC RT-PCR), a technique in which cDNA copies are made of the mRNA species within a single cell, including mRNA's of low abundance [9]. At the same time the amounts of the cDNAs produced can be kept in relative proportion to the original mRNA starting material, allowing for studies of relative changes in gene expression. However, a compromise for the proportional representation of the cDNAs is that the length of the resultant cDNAs is shortened to approximately 300–700 bp at the extreme 3′ end. Groups using the GSC RT-PCR technique have documented quantitative changes in expression during haematopoietic development 9, 10, 11and endochondral ossification [12]as well as phenotyping of mature osteoblasts [13].

We have been interested in using this technique to study genes involved in the process of metastasis [14], since this process involves individual cells which may represent only a small fraction of cells within a much larger population of tumor cells. These metastatic cells have an altered phenotype but because they are only a very small fraction within the tumor population, bulk methods are not sensitive enough to detect altered expression of metastasis-related genes in these cells. Prior to initiating these studies we examined the sensitivity of GSC RT-PCR for studies with single cells. In this manuscript we describe experiments which determined the amount of variation generated by the method itself, by examining the expression of specific genes in single cells or in single-cell-equivalent aliquots obtained from groups of cells.

Section snippets

GSC RT-PCR

Conditions for GSC RT-PCR have been described previously [9]. Briefly, individual cells were lysed in 4 μl of 1st strand buffer, consisting of 0.4 U Human Placental RNase Inhibitors (Pharmacia, Montreal, PQ) and 0.1 U of InhibitAce (5′–3′ Research, Boulder, CO) in 0.25 mM dNTPs, 0.13 OD/ml oligo dT₂₀, 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3.0 mM MgCl₂, and 0.5% NP-40, then heated to 65°C for 2 min and then allowed to cool at room temperature for 3 min. The mRNA was reverse transcribed with 2.0 U of

Results

Differences in gene expression between single cells were studied both in proliferating tumour cells (KHT-LP1, a murine fibrosarcoma [16]) and in stationary phase, early passage, normal rat embryo fibroblasts (REF), which arrest in G₀/G₁ of the cell cycle and do not cycle once they reach confluence [17]. Fig. 1 shows a representative phosphorimage for amplified samples of individual cells or single cell aliquots probed with L32 and GAPDH for these two cell lines. Fig. 2A and B present the

Discussion

The interpretation of gene expression at the single cell level using the GSC RT-PCR technique relies on information relating to the variation introduced by the method. Our initial observations (Fig. 2) from studying single cells derived from the same initial populations, suggested that there was a large amount of variation in the gene expression of `housekeeping' genes. To verify that the observed variation in the gene expression was real, we performed experiments where we used pooled starting

Acknowledgements

This work was supported by grants from the Medical Research Council of Canada and from the National Cancer Institute of Canada with funds raised by the Terry Fox Run. The first two authors (LHB+AJ) have contributed equally to the work described in this paper.

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Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR)

Abstract

Introduction

Section snippets

GSC RT-PCR

Results

Discussion

Acknowledgements

Trends in Biotechnology

Cell

Cell

Exp. Cell Res.

Int. J. Rad. Oncol. Biol. Phys.

Int. J. Rad. Oncol. Biol. Phys

Immunol Today

The clonal evolution of tumor cell populations

Science

A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas

Cancer Res.

In situ PCR: pathologist's dream or nightmare?

J. Pathol.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes

Proc. Natl. Acad. Sci. USA

Serial analysis of gene expression

Science