Absolute quantification by droplet digital PCR versus analog real-time PCR
Christopher Hindson,John R. Chevillet,Hilary A. Briggs,Emily N. Gallichotte,Ingrid K. Ruf,Benjamin J. Hindson,Robert L. Vessella,Muneesh Tewari +7 more
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TLDR
A comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision and improved day-to-day reproducibility of dd PCR but with comparable sensitivity, which translated to superior diagnostic performance for identifying individuals with cancer.Abstract:
Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.read more
Citations
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Quantitative and stoichiometric analysis of the microRNA content of exosomes.
John R. Chevillet,Qing Kang,Ingrid K. Ruf,Hilary A. Briggs,Lucia Vojtech,Sean M. Hughes,Heather H. Cheng,Jason D. Arroyo,Emily K. Meredith,Emily N. Gallichotte,Era L. Pogosova-Agadjanyan,Colm Morrissey,Derek L. Stirewalt,Florian Hladik,Evan Y. Yu,Celestia S. Higano,Muneesh Tewari +16 more
TL;DR: It is shown here that most exosomes derived from standard preparations do not harbor many copies of miRNA molecules, and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication.
Journal ArticleDOI
Methodological Guidelines to Study Extracellular Vesicles.
Frank A. W. Coumans,Alain Brisson,Edit I. Buzás,Françoise Dignat-George,Esther E.E. Drees,Samir El-Andaloussi,Samir El-Andaloussi,Costanza Emanueli,Costanza Emanueli,Aleksandra Gasecka,Aleksandra Gasecka,An Hendrix,Andrew F. Hill,Romaric Lacroix,Yi Lee,Ton G. van Leeuwen,Nigel Mackman,Imre Mäger,Imre Mäger,John P. Nolan,Edwin van der Pol,D. Michiel Pegtel,Susmita Sahoo,Pia Siljander,Guus Sturk,Olivier De Wever,Rienk Nieuwland +26 more
TL;DR: This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs.
Journal ArticleDOI
Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients.
Fengting Yu,Liting Yan,Nan Wang,Siyuan Yang,Linghang Wang,Yunxia Tang,Guiju Gao,Sa Wang,Chengjie Ma,Ruming Xie,Fang Wang,Chianru Tan,Lingxiang Zhu,Guo Yong,Fujie Zhang +14 more
TL;DR: Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.
Journal ArticleDOI
Liquid Biopsy for Advanced Non-Small Cell Lung Cancer (NSCLC): A Statement Paper from the IASLC
Christian Rolfo,Philip C. Mack,Giorgio V. Scagliotti,Paul Baas,Fabrice Barlesi,Trever G. Bivona,Roy S. Herbst,Tony Mok,Nir Peled,Robert Pirker,Luis E. Raez,Martin Reck,Jonathan W. Riess,Lecia V. Sequist,Frances A. Shepherd,Lynette M. Sholl,Daniel Shao-Weng Tan,Heather A. Wakelee,Ignacio I. Wistuba,Murry W. Wynes,David P. Carbone,Fred R. Hirsch,David R. Gandara +22 more
TL;DR: It is concluded that liquid biopsy approaches have significant potential to improve patient care, and immediate implementation in the clinic is justified in a number of therapeutic settings relevant to NSCLC.
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Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization
TL;DR: This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis, and discusses strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts.
References
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Jason D. Arroyo,John R. Chevillet,Evan M. Kroh,Ingrid K. Ruf,Colin C. Pritchard,Donald F. Gibson,Patrick S. Mitchell,Christopher F. Bennett,Era L. Pogosova-Agadjanyan,Derek L. Stirewalt,Jonathan F. Tait,Muneesh Tewari +11 more
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Journal ArticleDOI
High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number
Benjamin J. Hindson,Kevin D. Ness,Donald A. Masquelier,Phillip Belgrader,Nicholas J. Heredia,Anthony J. Makarewicz,Isaac J. Bright,Michael Y. Lucero,Amy L. Hiddessen,Tina C. Legler,Tyler K. Kitano,Michael R. Hodel,Jonathan Petersen,Paul Wyatt,Erin R. Steenblock,Pallavi Shah,Luc J. Bousse,Camille B. Troup,Jeffrey Clark Mellen,Dean K. Wittmann,Nicholas G. Erndt,Thomas H. Cauley,Ryan T. Koehler,Austin P. So,Simant Dube,Klint Rose,Luz Montesclaros,Shenglong Wang,David P. Stumbo,Shawn Hodges,Steven Romine,Fred P. Milanovich,Helen E. White,John F. Regan,George Karlin-Neumann,Christopher Hindson,Serge Saxonov,Bill W. Colston +37 more
TL;DR: A high-throughput droplet digital PCR system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow is described that will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.
Journal ArticleDOI
Quantification of mRNA using real-time RT-PCR
TL;DR: A series of RT-qPCR protocols are described that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible in molecular medicine, biotechnology, microbiology and diagnostics.
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