Massively parallel detection ofgene expression in single cells using subnanolitre wells

Abstract

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 directly from individual cells by one-step, single-cell, reverse transcription polymerase chain reaction (RT-PCR). Our approach permits the detection of mRNA transcripts of interest for more than 6000 single cells in parallel per assay with high sensitivity and specificity for constitutively active genes. This simple method was also combined with microengraving and image-based cytometry to examine the relationships between gene expression and cellular secretion of antibodies in a clonal population. We observed that most individual human B cell hybridomas transcribed a requisite gene for their antibodies, but only a subset of those cells secreted the antibody. The technique should also allow the detection of replicating intracellular pathogens such as retroviruses.