dPCR: A Technology Review

Quan, Phenix‐Lan; Sauzade, Martin; Brouzés, Eric

doi:10.3390/s18041271

Cited by 453 publications

(376 citation statements)

References 138 publications

(244 reference statements)

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“…Thresholds were then used to determine the number of positive and negative partitions. Positive counts were fitted to a Poisson distribution to determine copy number 23 . mtDNA-CN was represented as the ratio between the number of ND1 copies/µL and the number of RPPH1 copies/µl.…”

Section: Whole Genome Sequencing Data Was Generated At the Baylor Colmentioning

confidence: 99%

Evaluation of mitochondrial DNA copy number estimation techniques

Longchamps¹,

Castellani²,

Newcomb³

et al. 2019

Preprint

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Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy measure for mitochondrial function and has been associated with several aging-related diseases. Although quantitative real-time PCR (qPCR) is the current gold standard method for measuring mtDNA-CN, mtDNA-CN can also be measured from genotyping microarray probe intensities and DNA sequencing read counts. To conduct a comprehensive examination on the performance of these methods, we use known mtDNA-CN correlates (age, sex, white blood cell count, Duffy locus genotype, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence (WES) data across 1,085 participants from the Atherosclerosis Risk in Communities (ARIC) study and 3,489 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We observe mtDNA-CN derived from WGS data is significantly more associated with known correlates compared to all other methods (p < 0.001). Additionally, mtDNA-CN measured from WGS is on average more significantly associated with traits by 5.6 orders of magnitude and has effect size estimates 5.8 times more extreme than the current gold standard of qPCR. We further investigated the role of DNA extraction method on mtDNA-CN estimate reproducibility and found mtDNA-CN estimated from cell lysate is significantly less variable than traditional phenol-chloroform-isoamyl alcohol (p = 5.44x10 -4 ) and silica-based column selection (p = 2.82x10 -7 ). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed.Δ Ct values >3 standard deviations from the mean of the plate. Outlier replicates were identified and excluded for samples with a Δ Ct standard deviation >0.5. The sample was excluded if the Δ Ct standard deviation remained >0.5 after replicate removal. We

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Section: Whole Genome Sequencing Data Was Generated At the Baylor Colmentioning

confidence: 99%

Evaluation of mitochondrial DNA copy number estimation techniques

Longchamps¹,

Castellani²,

Newcomb³

et al. 2019

Preprint

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“…The level of novel aberrant transcripts detected in WT/KI are about 50% of the KI/KI mice, exhibiting a dosage-dependent expression of the mutant allele. As ddPCR quantification is based on binomial statistics (Quan, Sauzade, & Brouzes, 2018), we were able to directly compare the expression levels between different transcripts in the KI/KI mouse eye cup. The number of copies of the missense mRNA transcript (E13E14) is about twice that of the E13-AS transcript and seven times more than the E13-DEL transcript F I G U R E 8 RNA-seq analysis of mouse eyecup RNA (RPE-Choroid-Sclera).…”

Section: The C1430a>g Mutation Impairs Rpe65 Mrna Splicingmentioning

confidence: 99%

Aberrant RNA splicing is the major pathogenic effect in a knock‐in mouse model of the dominantly inherited c.1430A>G humanRPE65mutation

Furhang

Ray

et al. 2019

Human Mutation

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Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock‐in (KI) mouse model using CRISPR/Cas9‐mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

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“…Of note, in real‐time PCR the amplified product can be monitored in real time as it becomes amplified and an amplification (standard) curve is established. Furthermore, these curves demonstrate a relative estimate of a product amplified after each cycle (Quan et al, ).…”

Section: Digital Polymerase Chain Reactionmentioning

confidence: 99%

“…Of note, in real-time PCR the amplified product can be monitored in real time as it becomes amplified and an amplification (standard) curve is established. Furthermore, these curves demonstrate a relative estimate of a product amplified after each cycle (Quan et al, 2018). According to Lane, the Food and Drug Administration (FDA) established dPCR to be a proven technique in clinical diagnosis and drug identification, due to the highly efficient consistency and clarity of its results.…”

Section: Digital Polymerase Chain Reactionmentioning

confidence: 99%

The evolution of molecular diagnosis using digital polymerase chain reaction to detect cancer via cell‐free DNA and circulating tumor cells

Melo-Silva

Lucena

Hueneburg

2019

Cell Biology International

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Cancer is one of the most important causes of death worldwide. The onset of cancer may be initiated due to a variety of factors such as environment, genetics or even due to personal lifestyle choices. To counteract this tremendous increase, the demand for a new technology has risen. By this means, the use of digital polymerase chain reaction (dPCR) has been shown to be a promising methodology in the early detection of many types of cancers. Furthermore, several researchers confirmed that the use of tumor cell‐free DNA (cfDNA) and circulating tumor cells (CTC) in peripheral blood is essential in revealing an early prognosis of such diseases. Besides this, it was established that dPCR might be used in a much more efficient, accurate, and reliable manner to amplify a variety of genetic material up to the identification of mutations in hematological diseases. Therefore, this article demonstrates the differences between conventional PCR and dPCR as a molecular technique to detect the early onset of cancer. Furthermore, CTC and cfDNA were officially approved by the Food and Drug Administration as new biological biomarkers in cancer development and monitoring.

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dPCR: A Technology Review

Cited by 453 publications

References 138 publications

Evaluation of mitochondrial DNA copy number estimation techniques

Evaluation of mitochondrial DNA copy number estimation techniques

Aberrant RNA splicing is the major pathogenic effect in a knock‐in mouse model of the dominantly inherited c.1430A>G humanRPE65mutation

The evolution of molecular diagnosis using digital polymerase chain reaction to detect cancer via cell‐free DNA and circulating tumor cells

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