Quantification of Plasma miRNAs by Digital PCR for Cancer Diagnosis

Ma, Jie; Li, Ning; Guarnera, Maria; Jiang, Feng

doi:10.4137/bmi.s13154

Cited by 105 publications

(117 citation statements)

References 39 publications

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“…The dPCRgyrA sensitivity levels observed in this study are similar to those previously reported by Pholwat et (23). In the literature, the maximum sensitivity reported for dPCR is 0.001% (one mutant sequence for 100,000 wild-type sequences) (28)(29)(30). Such a high sensitivity may be obtained with a higher number of individual reactions (e.g., more than 1,000,000 individual reactions) and/or a different dPCR technique (droplet dPCR versus dPCR on a chip).…”

Section: Discussionsupporting

confidence: 81%

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

Hennebique

Bidart

Jarraud

et al. 2017

Antimicrob Agents Chemother

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The emergence of fluoroquinolone (FQ)-resistant mutants of Legionella pneumophila in infected humans was previously reported using a next-generation DNA sequencing (NGS) approach. This finding could explain part of the therapeutic failures observed in legionellosis patients treated with these antibiotics. The aim of this study was to develop digital PCR (dPCR) assays allowing rapid and accurate detection and quantification of these resistant mutants in respiratory samples, especially when the proportion of mutants in a wild-type background is low. We designed three dPCRgyrA assays to detect and differentiate the wild-type and one of the three gyrA mutations previously described as associated with FQ resistance in L. pneumophila: at positions 248C¡T (T83I), 259G¡A (D87N), and 259G¡C (D87H). To assess the performance of these assays, mixtures of FQ-resistant and -susceptible strains of L. pneumophila were analyzed, and the results were compared with those obtained with Sanger DNA sequencing and real-time quantitative PCR (qPCR) technologies. The dPCRgyrA assays were able to detect mutated gyrA sequences in the presence of wild-type sequences at up to 1:1,000 resistant/susceptible allele ratios. By comparison, Sanger DNA sequencing and qPCR were less sensitive, allowing the detection of gyrA mutants at up to 1:1 and 1:10 ratios, respectively. When testing 38 respiratory samples from 23 legionellosis patients (69.6% treated with an FQ), dPCRgyrA detected small amounts of gyrA mutants in four (10.5%) samples from three (13.0%) patients. These results demonstrate that dPCR is a highly sensitive alternative to quantify FQ resistance in L. pneumophila, and it could be used in clinical practice to detect patients that could be at higher risk of therapeutic failure.KEYWORDS digital PCR, antibiotic resistance, fluoroquinolones, gyrA, Legionella pneumophila L egionella pneumophila, a Gram-negative, facultative, intracellular bacterium, is the causative agent of legionellosis, a severe pneumonia associated with mortality rates ranging from 5% to 25% (1). The diagnosis of this disease mainly relies on a urinary antigen test and culture and PCR testing of respiratory samples. The first-line drugs for the treatment of legionellosis are the macrolides and the fluoroquinolones (FQ) (2). In vitro antibiotic susceptibility testing of Legionella species strains is currently not recommended on a routine basis, especially because no standardized method is available from the CLSI (Clinical and Laboratory Standards Institute) or the EUCAST (European Committee on Antimicrobial Susceptibility Testing). However, therapeutic failures and

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Section: Discussionsupporting

confidence: 81%

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

Hennebique

Bidart

Jarraud

et al. 2017

Antimicrob Agents Chemother

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“…The development of digital PCR will improve the performance of qRT-PCR, without the use of endogenous or exogenous controls to normalize the results. In contrast, digital PCR is a direct method for quantifying nucleic acids, particularly useful for samples with low abundant miRNAs, and showing a high degree of sensitivity and precision compared to qRT-PCR [17,18]. Finally, next generation sequencing (NGS) platforms, such as those from Solexa/Illumina and 454 Life Sciences/Roche, have been developed to find novel miRNAs, generating a complete expression profile, distinguishing sequentially similar miRNAs and identifying point mutations.…”

Section: Biogenesis and Function Of Mirnasmentioning

confidence: 99%

miRNAs as novel biomarkers in the management of prostate cancer

Filella

Foj

2017

Clinical Chemistry and Laboratory Medicine (CCLM)

100

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microRNAs (miRNAs) are small non-coding RNAs that control gene expression posttranscriptionally and are part of the giant non codifying genoma. Cumulating data suggest that miRNAs are promising potential biomarkers for many diseases, including cancer. Prostate cancer (PCa) detection is currently based in the serum prostate-specific antigen biomarker and digital rectal examination. However, these methods are limited by a low predictive value and the adverse consequences associated with overdiagnosis and overtreatment. New biomarkers that could be used for PCa detection and prognosis are still needed. Recent studies have demonstrated that aberrant expressions of microRNAs are associated with the underlying mechanisms of PCa. This review attempts to extensively summarize the current knowledge of miRNA expression patterns, as well as their targets and involvement in PCa pathogenesis. We focused our review in the value of circulating and urine miRNAs as biomarkers in PCa patients, highlighting the existing discrepancies between different studies, probably associated with the important methodological issues related to their quantitation and normalization. The majority of studies have been performed in serum or plasma, but urine obtained after prostate massage appears as a new way to explore the usefulness of miRNAs. Large screening studies to select a miRNA profile have been completed, but bioinformatics tools appear as a new approach to select miRNAs that are relevant in PCa development.Promising preliminary results were published concerning miR-141, miR-375 and miR-21, but larger and prospective studies using standardized methodology are necessary to define the value of miRNAs in the detection and prognosis of PCa.

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“…(2) It has shown increased sensitivity while detecting low target copies without the need for any pre-amplification steps. (3) It is relatively insensitive to potential PCR inhibitors [20][21][22][23].…”

Section: Ddpcr Is Highly Accurate In Circrna Quantificationmentioning

confidence: 99%

Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs

Chen

Zhang

Tan

et al. 2017

Biotechnology & Biotechnological Equipment

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Diagnostics based on circulating circular RNA (circRNA) is an emerging field for noninvasive molecular diagnosis, owing to the stable circular structure of circRNA. However, the uniquely circular structure still poses a challenge for circRNA quantification in the research community. Here, we verified the discrepancy in the circRNA quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR), which is caused by the rolling reverse transcription (RT) product originating from the circular structure. In addition, we detected the stability of cell-free circRNA in serum/ plasma and determined the pre-analysis sampling procedure that will compromise the quantification of circRNA. Our results showed that prolonged RT incubation time resulted in multiple PCR products from circular RNA, which will reduce the accuracy of circRNA quantification by RT-qPCR technology, whereas ddPCR can address this limitation and could be a good alternative to qPCR for circRNA quantification. CircRNA showed a high stability in promptly separated serum/ plasma but not in delay-separated samples.

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Quantification of Plasma miRNAs by Digital PCR for Cancer Diagnosis

Cited by 105 publications

References 39 publications

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

miRNAs as novel biomarkers in the management of prostate cancer

Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs

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