Massively parallel detection of gene 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.

High-throughput microfluidic single-cell RT-qPCR

“…Initial attempts to perform RT-qPCR in low nanoliter volumes were found to produce highly variable results, including nonspecific amplification and inconsistent detection of abundant transcripts (18). Cell lysate dilutions showed that reaction inhibition becomes significant at concentrations in excess of 0.2 cells∕nL, or 10 cells∕50 nL-reaction (Fig.…”

Integrated process design for single‐cell analytical technologies

“…Finally, these processes are nondestructive, which allows the recovery of viable cells for expansion in vitro, 32,33 or massively parallel detection of expressed mRNA transcripts. 34 The evaluation of the diversity in the humoral, or antibody, response raised in a model vaccination provides a representative problem to illustrate how these interchangeable operations can enable a comprehensive bioanalytical process. 35 We immunized mice with a prime-boost regime using ovalbumin-a model protein designed to mimic a recombinant immunogen in a vaccine-and then collected a series of data for a set of B cells using a combination of image-based cytometry and serial microengraving.…”

Microfluidics-Based Single-Cell Functional Proteomics for Fundamental and Applied Biomedical Applications

“…Both of these advantages are very hard to replicate using flow cytometry tools. An additional advantage of the microengraving platform is that it can be modified for PCR-based detection of a few transcripts at the single-cell level (69), although the combination of proteins and transcripts from the same single cells, using microengraved platforms, has not yet been reported. Figure 3 illustrates a microengraving experiment designed to detect antibody-secreting B cells collected from specific locations from a healthy mouse or a mouse model of the autoimmune disorder known as Sjögren's syndrome (68).…”