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Steps to achieve quantitative measurements of microRNA using two-step droplet digital PCR

Metadata Updated: July 29, 2022

Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real time PCR (qRT PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two step principles used for miRNA qRT PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

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Public: This dataset is intended for public access and use. License: See this page for license information.

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Dates

Metadata Created Date March 11, 2021
Metadata Updated Date July 29, 2022
Data Update Frequency irregular

Metadata Source

Harvested from NIST

Additional Metadata

Resource Type Dataset
Metadata Created Date March 11, 2021
Metadata Updated Date July 29, 2022
Publisher National Institute of Standards and Technology
Maintainer
Identifier 5BD6911D381AB2E3E0531A57068151FA1869
Language en
Data Last Modified 2017-10-19
Category Bioscience: Genomic measurements
Public Access Level public
Data Update Frequency irregular
Bureau Code 006:55
Metadata Context https://project-open-data.cio.gov/v1.1/schema/data.json
Schema Version https://project-open-data.cio.gov/v1.1/schema
Catalog Describedby https://project-open-data.cio.gov/v1.1/schema/catalog.json
Harvest Object Id b708536b-7ffa-4020-ade1-e8b0b787f14c
Harvest Source Id 74e175d9-66b3-4323-ac98-e2a90eeb93c0
Harvest Source Title NIST
License https://www.nist.gov/open/license
Program Code 006:045
Source Datajson Identifier True
Source Hash 510645a2351407b67a678419fbd59f97d56bc022
Source Schema Version 1.1

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