Quantification of multiple gene expression in individual cells.

@article{citeulike:381101, abstract = {Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading.}, address = {INSERM U591, Institut Necker, Paris, 75015 France.}, author = {Peixoto, A. and Monteiro, M. and Rocha, B. and Veiga-Fernandes, H. }, citeulike-article-id = {381101}, comment = {This paper proposes a new insight into cell function. This new developped method allows the evaluation of the expression of multiple genes in individual cells ex vivo.}, doi = {10.1101/gr.2890204}, issn = {1088-9051}, journal = {Genome Res}, keywords = {cell, single}, month = {October}, number = {10A}, pages = {1938--1947}, posted-at = {2005-11-04 22:57:55}, priority = {0}, title = {Quantification of multiple gene expression in individual cells.}, url = {http://dx.doi.org/10.1101/gr.2890204}, volume = {14}, year = {2004} }

TY - JOUR ID - citeulike:381101 L3 - citeulike-article-id:381101 TI - Quantification of multiple gene expression in individual cells. JF - Genome Res VL - 14 IS - 10A SP - 1938 EP - 1947 AD - INSERM U591, Institut Necker, Paris, 75015 France. SN - 1088-9051 N2 - Quantitative gene expression analysis aims to define the gene expression patterns determining cell behavior. So far, these assessments can only be performed at the population level. Therefore, they determine the average gene expression within a population, overlooking possible cell-to-cell heterogeneity that could lead to different cell behaviors/cell fates. Understanding individual cell behavior requires multiple gene expression analyses of single cells, and may be fundamental for the understanding of all types of biological events and/or differentiation processes. We here describe a new reverse transcription-polymerase chain reaction (RT-PCR) approach allowing the simultaneous quantification of the expression of 20 genes in the same single cell. This method has broad application, in different species and any type of gene combination. RT efficiency is evaluated. Uniform and maximized amplification conditions for all genes are provided. Abundance relationships are maintained, allowing the precise quantification of the absolute number of mRNA molecules per cell, ranging from 2 to 1.28 x 10(9) for each individual gene. We evaluated the impact of this approach on functional genetic read-outs by studying an apparently homogeneous population (monoclonal T cells recovered 4 d after antigen stimulation), using either this method or conventional real-time RT-PCR. Single-cell studies revealed considerable cell-to-cell variation: All T cells did not express all individual genes. Gene coexpression patterns were very heterogeneous. mRNA copy numbers varied between different transcripts and in different cells. As a consequence, this single-cell assay introduces new and fundamental information regarding functional genomic read-outs. By comparison, we also show that conventional quantitative assays determining population averages supply insufficient information, and may even be highly misleading. KW - cell KW - single N1 - This paper proposes a new insight into cell function. This new developped method allows the evaluation of the expression of multiple genes in individual cells ex vivo. AU - Peixoto, A AU - Monteiro, M AU - Rocha, B AU - Veiga-Fernandes, H PY - 2004/10// UR - http://dx.doi.org/10.1101/gr.2890204 ER -