Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context.

No abstract

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one well-studied aspect of epigenetic regulation, but is challenging to quantify accurately. In this study, we introduce a digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation. Combining the efficiency of methylation-sensitive restriction enzymes with the quantitative power of digital PCR, DNA methylation is measured accurately without the need to treat the DNA samples with sodium bisulphite. Moreover, as the combination of PCR amplicon and restriction enzyme is flexible, the context and density of DNA methylation can be taken into account. Additionally, by extending the experimental setup to a multiplex digital PCR, methylation markers may be analysed together with physically linked genetic markers to determine the allele-specificity of the methylation. In-silico simulations demonstrated the mathematical validity of the experimental setup. Next the approach was validated in a variety of healthy and malignant reference samples in the context of RASSF1A promotor methylation. RASSF1A is an established tumour suppressor gene, that is aberrantly methylated in many human cancers. A dilution series of well-characterized reference samples cross-validated the sensitivity and dynamic range of the approach. Compared to conventional PCR based methods, digital PCR provides a more accurate and more sensitive approach to quantify DNA methylation. As no sodium bisulphite conversion is needed, also analysis of minute amounts of DNA could be carried out efficiently.