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

Abstract

Background Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging. In particular, nucleic acid extraction for quantification of gene targets may lead to target loss. Here, we report the development and validation of a novel crude lysate ddPCR assay for the absolute quantification of rare genes, TRECs in our case, from infrequent cells, that removes the need for DNA extraction, hence minimizing the target loss. Methods The analytical validation was performed on PBMCs extracted from the blood of healthy donors. Standard ddPCR was first optimized to detect TREC copies/cell and then applied to a crude lysate ddPCR assay. The assay was optimized by varying several steps. The optimised assay was directly compared to standard ddPCR and the performance of the assay quantified. Results The newly developed assay showed good agreement with the standard ddPCR assay in the range from 0.0003 to 0.01 TRECs/cell. The assay had a limit of quantification of <0.0003 TRECs/cell and a limit of detection of <0.0001 TRECs/cell; this performance is favourable compared to standard ddPCR. The intra-assay variation was low. This method can also be applied to fixed and permeabilized cells. Conclusions The newly developed crude lysate ddPCR assay for quantifying rare targets from limited samples has high accuracy, specificity, and reproducibility; additionally, it eliminates the need for DNA extraction for absolute quantification. The assay has the potential to be used for quantification of other trace targets from small samples.