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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...
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Updated: Jan 15, 2026

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
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Multiplexed Digital PCR Reference Gene Measurement for Genomic and Cell-Free DNA Analysis.

Dilek Yener1,2, Eloise J Busby1, Jo Vandesompele3

  • 1National Measurement Laboratory, LGC, Guildford GU2 7XY, UK.

Cells
|October 15, 2025
PubMed
Summary
This summary is machine-generated.

A new digital PCR (dPCR) method using a pentaplex reference gene panel offers a robust solution for total DNA quantification. This approach improves accuracy in precision medicine by providing lower measurement uncertainty for genomic DNA and cell-free DNA.

Keywords:
dPCRgenome equivalentsliquid biopsyprecision medicinereference gene stability

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Area of Science:

  • Biochemistry
  • Genomics
  • Molecular Biology

Background:

  • Accurate DNA quantification is crucial for precision medicine and somatic variant detection.
  • Existing methods for total DNA quantification lack a standardized gold standard.
  • Genomic DNA (gDNA) and cell-free DNA (cfDNA) are key inputs for genomic workflows.

Purpose of the Study:

  • To develop and validate a pentaplex reference gene panel using digital PCR (dPCR) as a candidate gold standard for total DNA quantification.
  • To compare the performance of a multiplex dPCR approach with two assay chemistries using gDNA and cfDNA.
  • To assess the method's suitability for copy number variation (CNV) quantification in cancer samples.

Main Methods:

  • Development of a pentaplex reference gene panel for dPCR.
  • Application of the panel to healthy donor gDNA and plasma cfDNA.
  • Measurement of ERBB2 (HER2) copy number variation in cancer cell line DNA.
  • Comparison of two dPCR assay chemistries for multiplex analysis.

Main Results:

  • The multiplex dPCR approach demonstrated robust performance and comparable results across two assay chemistries with a wide dynamic range.
  • Reference gene ratios were near 1:1 in healthy samples, with minor variations (<1.2-fold) in one target.
  • Expanded relative measurement uncertainty ranged from 12.1-19.8% for healthy gDNA and 9.2-25.2% for cfDNA.
  • The multiplex method yielded lower measurement uncertainty than single-reference methods for total DNA quantification.

Conclusions:

  • The developed pentaplex dPCR panel serves as a reliable candidate reference method for total DNA quantification.
  • The multiplex approach offers improved accuracy and reduced measurement uncertainty, beneficial for calibration in genomic workflows.
  • This method mitigates potential biases in CNV quantification for cancer samples, addressing genome instability challenges.