The relationship between the expression of particular genes in cells and their impact on phenotypic characteristics is important for understanding how cells regulate responses to their environment.  We have developed a microwell-based method to detect copies of mRNA transcripts among individual cells by single-cell, reverse transcription polymerase chain reaction (RT-PCR).  This approach permits parallel detection of one or more mRNA transcripts of interest for more than 10,000 cells per assay. The assay exhibited high sensitivity and specificity for constitutively active genes, and can be multiplexed using distinct fluorescent probes for different genes of interest.  This simple method can also be combined with microengraving and imaging cytometry to examine the relationships between the gene expression and the cellular secretion of antibodies in peripheral blood mononuclear cells.  The technique should also allow the detection of replicating intracellular pathogens such as retroviruses.

Massively parallel detection of gene expression in single cells using subnanoliter wells and multiplexed RT-PCR

240th National Meeting (2010)

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