A high level of heterogeneity in gene expression profiles is observed among cells within a tissue or a cell population. Even cells with the same apparent phenotype can display wide variations in the set of expressed genes and their expression levels.
Single-cell analysis can be used to study the profiles of different cell types within a tissue, variations in cells within a population, and differences in cellular processes such as differentiation or responses to stimuli. Due to the small amount of nucleic acid present in one cell, the use of qPCR for single-cell assays is technically challenging. Digital PCR, with its increased sensitivity and absolute quantification, can overcome some of the barriers to single-cell analysis.
In the Droplet Digital™ PCR (ddPCR™) System, each PCR sample is partitioned into a large number of nanodroplets prior to amplification. After amplification, fluorescence in any droplet indicates the amplification of the target sequence. Droplets are thus classified as either positive or negative, yielding digital (binary) data. Absolute quantification is then achieved using Poisson statistical analysis.
ddPCR technology has several advantages over other methods for single-cell analysis:
An early study demonstrating the potential of digital PCR in single-cell analysis involved the characterization of hematopoietic progenitor cells (Warren et al. 2006). The authors investigated transcription factor expression in cells from several classes of hematopoietic precursors and demonstrated that there were high levels of heterogeneity in the expression of transcription factors among individual cells.
In addition to using digital PCR to study gene expression in individual cells, ddPCR technology can be used to generate a single-cell library for further analysis by techniques such as next-generation sequencing (Tewhey et al. 2009). Minimizing the bias against low-abundance transcripts enables generation of deep sequencing libraries with broader coverage.
Although single-cell analysis can be extremely challenging, digital PCR can be employed to overcome some of the technical problems of single-cell qPCR assays. The sensitivity of the ddPCR System can facilitate expanded analyses of single cells, the characterization of differences among cells, and changes in cells over time. ddPCR technology is making single-cell studies more sensitive and more precise.
Tewhey R et al. (2009). Microdroplet-based PCR amplification for large scale targeted sequencing. Nat Biotechnol 27 (11):1,025-31. [Erratum: Nat Biotechnol 2010 28:178]. PMID: 19881494
Warren L et al. (2006). Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Acad Nat Sci USA 103:17, 807–17, 812. PMID: 17098862
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