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Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

Targeted therapies in many cancers have allowed unprecedented progress in the treatment of disease. However, routine implementation of genomic testing is constrained due to: 1) limited amounts of sample (pg–ng range) per biological specimen, 2) diagnostic turnaround time and workflow, 3) cost, and 4) difficulties in detection of mutational loads below 5%. KRAS is mutated in approximately 40% of colorectal cancers (CRCs). The majority of mutations affect codons 12, 13, and 61 and indicate a negative response to anti–epidermal growth factor receptor (EGFR) therapy. To optimize therapy strategies for personalized care, it is critical to rapidly screen patient samples for the presence of multiple KRAS mutations.

The variant histone H2AX is phosphorylated in response to UV irradiation of primary human fibroblasts in a complex fashion that is radically different from that commonly reported after DNA double-strand breaks. H2AX phosphorylation after exposure to ionizing radiation produces foci, which are detectable by immunofluorescence microscopy and have been adopted as clear and consistent quantitative markers for DNA double-strand breaks. Here we show that in contrast to ionizing radiation, UV irradiation mainly induces H2AX phosphorylation as a diffuse, even, pan-nuclear staining. UV induced pan-nuclear phosphorylation of H2AX is present in all phases of the cell cycle and is highest in S phase. H2AX phosphorylation in G 1 cells depends on nucleotide excision repair factors that may expose the S-139 site to kinase activity, is not due to DNA double-strand breaks, and plays a larger role in UV-induced signal transduction than previously realized.

DNA damage-induced cell-cycle "checkpoint" responses reduce the mutagenic effects of this damage. However, the maintenance of genomic stability comes at a price: the slowing of growth and a delay in the development of critical tissues. In mammals, every mutated cell has the potential to become cancerous and therefore lethal. In plants, the risk of lethal cancers is essentially nil and the costs of delays in development are very high. Here, we investigate DNA damage checkpoint responses in meristematic (root and shoot tip) versus strictly somatic (stomatal and endoreduplicating) tissues in plants. We find that the ionizing radiation (IR)-induced cell-cycle responses observed in the root and shoot tip meristems do not apply to more differentiated tissues.

The recognition that tumors and other apparently homogeneous cell populations are often heterogeneous and display stochastic expression of their genes, has motivated the development of methods to profile gene expression in single cells. Among these, RNA-Seq is the most powerful for broad-scale profiling and discovery of genes and pathways influencing biological processes and states of interest. However, given the extent of sample manipulation required to prepare each RNA-Seq library – and the trade-off between sensitivity and sample throughput – it is important to validate discovery findings by orthogonal digital methods with comparable or greater sensitivity and accuracy. Droplet Digital PCR is such a method that can rapidly and cost-effectively replicate (or challenge) RNA-Seq results with minimal sample processing when defined targets are to be verified (Chen et al, 2012). Furthermore, ddPCR is performed in 96-well plates and is well-suited to high throughput studies of focused sets of genes in large numbers of single cells (e.g. hundreds in a day). Here we present a simple and robust workflow for profiling multiplexed, transcript targets in flow-sorted, Jurkat single-cells using Taqman 5’ probe hydrolysis assays. We demonstrate that Bio-Rad's QX100 & QX200 Droplet Digital PCR systems provide absolute counts of transcripts from >100,000 copies to More than 30 cell-cycle genes have been evaluated by ddPCR in exponentially growing Jurkat cells. Several genes, including CCNB1, CDK1 and DNM2, show an unusually broad range of transcript levels (over 2 logs), suggestive of their known phase-specific cell-cycle roles. Jurkat cells have been FACS sorted for either G0/G1 (“G1”) or G2/M (“G2”) single-cells using DyeCycle Orange to stain for DNA content, and are being assessed for phase-specific expression profiles. Preliminary results indicate that G2 single-cells are enriched for higher CCNB1 and CDK1 transcript levels as expected from their role in promoting early events in mitosis; however, even G2 cells still show expression levels spanning ∼2 logs, suggesting cell-cycle phase heterogeneity and/or bursty transcription within the sorted G2 population. While the average mRNA level of even the house-keeping gene, GAPDH, is several-fold higher in G2 cells as compared to G1 cells, transcripts for CCNB1 and CDK1 average ∼6-fold higher in G2 than in G1. In this work, we demonstrate both the analytical performance and the biological utility of droplet digital PCR for single-cell analysis. This same workflow could be used for fingerprinting and study of sub-populations in tumors. Citation Format: George Karlin-Neumann, Shenglong Wang, Camille Troup, Yann Jouvenot, Eli Hefner. Rapid and ultra-sensitive single-cell transcript profiling with droplet digital PCR (ddPCR): Application to cell cycle analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2014-3491

Targeted therapies in many forms of cancer today have allowed unprecedented progress in the treatment of disease. Despite these advances, routine implementation of genomic testing is still limited by: 1) methods to detect, with confidence, mutational loads below 10%, 2) limited amounts of sample (pg-ng range) per biological specimen, 3) diagnostic turnaround time, and 4) cost. In metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor antibodies (αEGFR) are used to target the wild-type EGFR receptor. However, KRAS is mutated in approximately 40% of colorectal cancers and is indicative of a negative response to αEGFR therapy. BRAF and PIK3CA mutations are also associated with poor response in the remaining patients with KRAS wild-type genotype. To optimize therapy strategies for personalized care, it is therefore critical to rapidly screen patient samples during the course of disease for the presence of multiple mutations. The low abundance of mutants and limited amount and quality of available clinical samples (FFPE and cfDNA) render it difficult to reliably detect multiple mutations with current platforms and methods. We have developed a multiplexing strategy for screening clinically-actionable KRAS, BRAF, and PIK3CA mutations in mCRC clinical samples using digital PCR. No pre-amplification step was required. This sensitive and inexpensive method reduces the risk of contamination and can be easily implemented in molecular diagnostic laboratories for rapid, routine screening and monitoring of residual disease in cancer patients. Citation Format: Wei Yang, Dawne N. Shelton, Samantha Cooper, Jennifer Berman, Svilen Tzonev, Eli Hefner, John F. Regan. Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2014-LB-115