Absolute quantification by droplet digital PCR versus analog real-time PCR

Hindson, Christopher M.; Chevillet, John R.; Briggs, Hilary; Gallichotte, Emily N.; Ruf, Ingrid K.; Hindson, Benjamin J.; Vessella, Robert L.; Tewari, Muneesh

doi:10.1038/nmeth.2633

Cited by 1,249 publications

(1,046 citation statements)

References 19 publications

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“…Dramatic reductions in day-to-day reproducibility (by a factor of seven) were also reported in comparing ddPCR and qPCR for assay of serum microRNA quantitation. 33 Ultimately, the validity of methylation assays of immune cells is determined by comparison with gold standard assays such as FACS. Figure 2 compares the mean T cell proportions determined by FACS, ddPCR, and qPCR.…”

Section: Resultsmentioning

confidence: 99%

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

et al. 2014

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Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

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Section: Resultsmentioning

confidence: 99%

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

et al. 2014

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“…The tissues from one little free-tailed bat from 2011 which yielded an isolate of ENTV, were flavivirus RNA-positive [16]. Development of specific assays targeting sub-genomic flaviviral RNA (sfRNA), which can be present in high amounts in infected cells, may be one approach to deciphering ambiguous serological results in future investigations [18,19]. …”

Section: Discussionmentioning

confidence: 99%

Neutralizing antibodies against flaviviruses, Babanki virus, and Rift Valley fever virus in Ugandan bats

Kading

Kityo

Mossel

et al. 2018

Infection Ecology & Epidemiology

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Introduction: A number of arboviruses have previously been isolated from naturally-infected East African bats, however the role of bats in arbovirus maintenance is poorly understood. The aim of this study was to investigate the exposure history of Ugandan bats to a panel of arboviruses. Materials and methods: Insectivorous and fruit bats were captured from multiple locations throughout Uganda during 2009 and 2011–2013. All serum samples were tested for neutralizing antibodies against West Nile virus (WNV), yellow fever virus (YFV), dengue 2 virus (DENV-2), Zika virus (ZIKV), Babanki virus (BBKV), and Rift Valley fever virus (RVFV) by plaque reduction neutralization test (PRNT). Sera from up to 626 bats were screened for antibodies against each virus. Results and Discussion: Key findings include the presence of neutralizing antibodies against RVFV in 5/52 (9.6%) of little epauletted fruit bats (Epomophorus labiatus) captured from Kawuku and 3/54 (5.6%) Egyptian rousette bats from Kasokero cave. Antibodies reactive to flaviviruses were widespread across bat taxa and sampling locations. Conclusion: The data presented demonstrate the widespread exposure of bats in Uganda to arboviruses, and highlight particular virus-bat associations that warrant further investigation.

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“…The technological advance in recent years enables us to quantify the ctDNA and to qualify its mutations [4]. Droplet digital PCR (ddPCR) is a new technique to quantify the number of target DNA molecules in the reaction tubes [5]. ddPCR is also highly sensitive to detect the rare mutated allele in the background of wild-type allele.…”

Section: Introductionmentioning

confidence: 99%

Detection of the <i>PIK3CA</i> Mutation in Circulating Tumor DNA as a Possible Predictive Indicator for Poor Prognosis of Early-Stage Breast Cancer

Sato¹,

Tanabe²,

Tsuboi³

et al. 2018

JCT

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Objectives: Circulating tumor DNA (ctDNA) is shown to provide the real-time genomic information of metastatic breast cancer. This study elucidates the clinico-pathological significance of ctDNA in early-stage breast cancer using the PIK3CA mutation as an indicator. Materials and Methods: Twenty-seven primary breast cancers without metastasis were surgically resected and pathologically diagnosed at the University of Tokyo Hospital, Japan. Genomic DNA of primary tumor was extracted from formalin-fixed and paraffin-embedded specimens. ctDNA was extracted from fresh-frozen plasma from patients. The PIK3CA mutations at E542K, E545K and H1047R were examined by Sanger sequencing or droplet digital PCR in 27 tumors and pre-and post-surgery plasma. Results: The PIK3CA mutations were detected in 13 (48%) of 27 primary tumors. These mutations did not significantly correlate with specific clinico-pathological characteristics of tumors. When ctDNA was examined, 4 (33%) of 12 cases carrying the mutated PIK3CA showed the identical mutation in pre-surgery plasma and 2 (50%) of them showed the identical mutations in post-surgery plasma. Interestingly, in these 2 cases in pathological stages IIIA and IA, fractional abundance of the mutated PIK3CA alleles to the total alleles in pre-surgery ctDNA was around 1% or more and was higher than that of the other two cases without PIK3CA mutations in post-surgery ctDNA. Conclusions: The PIK3CA mutation in ctDNA is detectable even in a subset of early-stage breast cancer. Furthermore, fractional abundance of the mutated PIK3CA in pre-surgery ctDNA could provide a possible predictive indicator for tumor burden and for choosing the appropriate adjuvant treatment of breast cancer.

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Absolute quantification by droplet digital PCR versus analog real-time PCR

Cited by 1,249 publications

References 19 publications

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Neutralizing antibodies against flaviviruses, Babanki virus, and Rift Valley fever virus in Ugandan bats

Detection of the <i>PIK3CA</i> Mutation in Circulating Tumor DNA as a Possible Predictive Indicator for Poor Prognosis of Early-Stage Breast Cancer

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Absolute quantification by droplet digital PCR versus analog real-time PCR

Cited by 1,249 publications

References 19 publications

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Neutralizing antibodies against flaviviruses, Babanki virus, and Rift Valley fever virus in Ugandan bats

Detection of the &lt;i&gt;PIK3CA&lt;/i&gt; Mutation in Circulating Tumor DNA as a Possible Predictive Indicator for Poor Prognosis of Early-Stage Breast Cancer

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Detection of the <i>PIK3CA</i> Mutation in Circulating Tumor DNA as a Possible Predictive Indicator for Poor Prognosis of Early-Stage Breast Cancer