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.
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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:
- Absolute quantification — in qPCR, amplified targets must be compared to a standard, which results in a relative quantification of the targets. ddPCR provides absolute quantification, which allows for direct comparison between targets
- Reduced sensitivity to PCR inhibition — since ddPCR data is collected at end-point (after 40 amplification cycles), it is not affected by collection point. This allows for increased tolerance to any factors reducing PCR efficiency
- Increased detection sensitivity for low-abundance transcripts — target sequence(s) will be present in only a subset of the nanoliter-sized droplets. Therefore, unlike single-tube PCR, each low-abundance transcript is segregated from the much larger pool of high-abundance transcripts. Decreased competition for primers increases the amplification and detection of rare transcripts. Thus, ddPCR technology can reduce the biases for overrepresentation of abundant sequences and underrepresentation of low-abundance transcripts observed in qPCR
- One-step RT-PCR is compatible with digital PCR — in single-cell studies, fewer manipulations are better for sample integrity and reduction of bias
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.
References
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