Kaur, C;
Adams, S;
Kibirige, CN;
Asquith, B;
(2025)
Absolute quantification of rare gene targets in limited samples using crude lysate and ddPCR.
Scientific Reports
, 15
, Article 9744. 10.1038/s41598-025-94115-w.
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Abstract
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.
| Type: | Article |
|---|---|
| Title: | Absolute quantification of rare gene targets in limited samples using crude lysate and ddPCR |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41598-025-94115-w |
| Publisher version: | https://doi.org/10.1038/s41598-025-94115-w |
| Language: | English |
| Additional information: | This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
| Keywords: | Crude lysate, Limited sample, Lysate buffer, Rare targets, TRECs, ddPCR, Humans, DNA, Polymerase Chain Reaction, Reproducibility of Results, Leukocytes, Mononuclear |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10215483 |
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