Sesame Street's Count von Count taught many of us the simple art of enumeration. However, most techniques used to quantify gene expression require more complicated mathematic algorithms. Now Warren et al. report a quantitative PCR technique that allows researchers to count the number of gene copies present in each cell in a recent article in Proceedings of the National Academy of Sciences USA.
In tissue samples with mixed cell types, gene expression changes in one cell type can obscure changes in another. Therefore, single-cell techniques are important in profiling cell-specific gene expression. However, many quantitative techniques require more RNA than is available from a single cell.
Because quantitative PCR expands signals exponentially, it is a good choice for quantification of gene expression in a single cell. Traditional quantitative PCR incorporates a fluorescent indicator dye into PCR products. Researchers quantify gene expression based on the PCR cycle at which fluorescence crosses a preset threshold. A sample with more PCR product will reach the threshold in fewer PCR cycles. To control for samples differences, researchers must normalize their data, but there is little agreement about how to do so.
The authors developed a 'digital PCR' technique that determines the absolute expression of gene copy numbers per cell and does not require normalization. They separated cells by flow cytometry and lysed them in buffer. Following reverse transcription, they loaded reactions under pressure into 12-welled chips. Then a membrane clamped each well into 1200 isolated compartments, and the chip was thermocycled. So, in all, each chip held 14,400 PCR reactions. For most genes with fewer than 1200 copies per cell, the authors quantified cellular gene expression by counting the number of positive PCR reactions among the 1200 reaction chambers.
The authors examined the expression of transcription factors in different cell types resulting from hematopoietic stem cell differentiation. They quantified expression of the metabolic gene GAPDH, which is commonly used in quantitative PCR to normalize gene expression. GAPDH expression varied nearly two-fold in the cell types examined, suggesting that it may be inappropriate for data normalization. Digital PCR does not require normalization and therefore avoids this complication.
Single-cell techniques are important in quantifying gene expression in the brain. Most brain regions contain both neurons and glia, and neighboring neurons are frequently genotypically and phenotypically different. Because development and some neural messengers regulate metabolic genes commonly used for normalization, digital PCR may be particularly useful in studies of genes involved in neuronal differentiation.