Absolute quantification of lung cancer related microRNA by droplet digital PCR

Wang, Ping; Fu, Jing; Li, Gang; Wu, Zhenhua; Cheng, Zule; Zhang, Jishen; Zhang, Honglian; Jia, Chunping; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

doi:10.1016/j.bios.2015.07.048

Cited by 90 publications

(63 citation statements)

References 42 publications

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“…It has been shown for various applications that ddPCR is capable of rare target DNA quantification with higher precision and accuracy compared to quantitative PCR [27, 37–39]. Although we did not perform quantitative PCR we found relative quantification measurements of MT copies down to 0.01%.…”

Section: Discussionmentioning

confidence: 69%

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

Ginkel

Huibers

et al. 2017

BMC Cancer

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BackgroundDuring posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR).Methods TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.ResultsIn all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2–422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667–156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%.ConclusionsOur results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3424-0) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 69%

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

Ginkel

Huibers

et al. 2017

BMC Cancer

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“…ddPCR can be used to detect rare mutations, can quantify gene expression with very low copy numbers, and can be multiplexed for enhanced efficiency . When applied to biomarker analysis of individuals with cancer, ddPCR has demonstrated superior diagnostic performance compared with other available molecular techniques . To our knowledge, the use of ddPCR technology has not yet been reported for the detection of oncogenic HPV in head and neck squamous cell carcinoma (SCC).…”

Section: Introductionmentioning

confidence: 99%

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

Biron

Kostiuk

Isaac

et al. 2016

Cancer

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BACKGROUND:The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV-OPSCC) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV-16 E6 and E7 oncoproteins or by cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultrasensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV-16 E6 and E7 in HPV-OPSCC. METHODS: Participants were recruited from January 2015-November 2015 at initial presentation to the University of Alberta Head and Neck Oncology Clinic. RNA was extracted, purified and quantified from prospectively collected participant tissues, and ddPCR was performed with fluorescent probes detecting HPV-16 E6 and E7. Results from ddPCR were compared with p16 IHC performed by clinical pathology as standard of care. RESULTS: Head and neck tissues were prospectively obtained from 68 participants including 29 patients with OPSCC, 29 patients with non-OPSCC and 10 patients without carcinoma. 79.2% of patients with OPSCC were p16 positive. The sensitivity and specificity of ddPCR HPV E6/E7 compared with p16 IHC in OPSCC was 91.3 and 100%, respectively. The amount of target RNA used was 1 ng, 20-50 times lower than reported by other for RT-qPCR HPV E6/E7. CONCLUSIONS: The ddPCR of HPV E6/E7 is a novel and highly specific method of detecting HPV-16 in OPSCC.

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“…Typically, qPCR dynamic range is 10 2 -10 7 copies of an individual cDNA. 47 DNA microarrays consist of an ordered arrangement of thousands of identified sequenced genes attached to a solid support. Each identified sequenced gene corresponds to a fragment of genomic DNA, cDNAs, PCR products, or chemically synthetic oligonucleotides, up to 70 mers.…”

Section: Challenges Faced By the Main Technologies In Nucleic Acmentioning

confidence: 99%

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

et al. 2016

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Nucleic acid biomarkers have enormous potential in non-invasive diagnostics and disease management. In medical research and in the near future in the clinics, there is a great demand for accurate miRNA, mRNA, and ctDNA identification and profiling. They may lead to screening of early stage cancer that is not detectable by tissue biopsy or imaging. Moreover, because their cost is low and they are non-invasive, they can become a regular screening test during annual checkups or allow a dynamic treatment program that adjusts its drug and dosage frequently. We briefly review a few existing viral and endogenous RNA assays that have been approved by the Federal Drug Administration. These tests are based on the main nucleic acid detection technologies, namely, quantitative reverse transcription polymerase chain reaction (PCR), microarrays, and next-generation sequencing. Several of the challenges that these three technologies still face regarding the quantitative measurement of a panel of nucleic acids are outlined. Finally, we review a cluster of microfluidic technologies from our group with potential for point-of-care nucleic acid quantification without nucleic acid amplification, designed to overcome specific limitations of current technologies. We suggest that integration of these technologies in a modular design can offer a low-cost, robust, and yet sensitive/selective platform for a variety of precision medicine applications.

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Absolute quantification of lung cancer related microRNA by droplet digital PCR

Cited by 90 publications

References 42 publications

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

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