Single Cell Transcript Quantification by Digital PCR
There is increasing evidence that pluripotent embryonic stem cell cultures (ESCs), once thought to be a uniform cell type, are in fact highly heterogeneous, especially in terms of RNA expression. Researchers at the University of Connecticut have confirmed that the expression of key developmental control genes in ESCs is highly heterogeneous and that analyzing individual stem cells is required for understanding this cellular complexity in stem cell biology.
Single cell expression analysis also has great potential for research involving microRNA. Until recently, understanding the dynamics of microRNA and mRNA expression within a cell population has been limited by the lack of an efficient and effective means to assay expression within individual cells. Digital PCR has been used to quantify the number of transcripts present in each cell, providing an unprecedented level of resolution to single-cell transcript analysis.
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Digital PCR results for GAPDH and Nanog detected on TaqMan® OpenArray® Digital PCR Plates
For analysis of Nanog expression in mouse ESCs by digital PCR, ESCs were sorted using FACS and deposited directly into lysis buffer from the Ambion® Single Cell-to-CT® Kit. In order to show the linear relationship between transcript level and number of cells, a range of cell quantities (20, 10, 5 , 3 and 1) were analyzed. After lysis, RNA was reversed transcribed and the entire RT reaction was amplified using GAPDH and Nanog TaqMan® assays in an OpenArray® Digital PCR Plate.
Digital PCR results for GAPDH and Nanog detected on TaqMan® OpenArray® Digital PCR Plates. (top panel right) Samples containing the indicated number of cells were analyzed on 8 subarrays, each with 64 reactions, for a total of 512 reactions per sample. Ct values for reactions with detected amplification are represented as a gradient from red (highly expressed) to black (not detected). The number of reactions in which amplification occurred was used to quantify the number of transcripts detected per cell. (top panel left) Scatter plot depicting the strong linear relationship between transcript quantity and the number of cells per sample for both GAPDH and Nanog. (r2 = .90). (bottom panel) Detection of GAPDH and Nanog transcripts in 2 individual cells. As expected, GAPDH is expressed at considerably higher levels than Nanog and its expression is comparable in both cells.