Absolute quantification of rare gene targets in limited samples using crude lysate and ddPCR

Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging, especially due to nucleic acid extraction. Using the commercially available genomic DNA (gDNA) extraction kits, which mostly include a DNA purification step through silica columns, magnetic beads or ethanol precipitation, are the preferred choice for many researchers. These kits, however, have a minimum cell number requirement for optimal DNA quality and yield. They are not ideal for use for clinical samples with limited cell numbers. Here, we report the development and validation of a novel crude lysate method for preparing DNA for the absolute quantification of rare genes, TRECs in our case, by droplet digital PCR (ddPCR), from infrequent cells, that removes the need for DNA extraction. Multiple optimization steps and analytical validation of this novel assay was performed on PBMCs extracted from the blood of healthy donors. The newly developed assay shows good agreement with standard ddPCR and has high accuracy, specificity, and reproducibility; additionally, it can also be applied to fixed and permeabilized cells. The assay has the potential to be used for quantification of other trace targets from limited cell samples.