Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease
July 3rd, 2018
Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.
This method may help answer key questions in the diagnostic field, such as whether micro RNAs are valid biomarkers in cardiovascular disease. Compared to quantitative real-time PCR, digital PCR exhibits superior technical qualities for quantifying microRNAs in the circulation. As the sample is divided into about 20, 000 droplets, no duplicate measurements are necessary, and no standard curve is needed for quantification.
Although this method may provide insight into quantification of microRNAs in cardiovascular patients, it may also be applied to other patients and diseases, as other microRNAs have shown promise as biomarkers in cancer progression, it may also be applied to oncological patients. Generally, individuals new to this method will struggle because the generated droplets are fragile and need to be handled with care. After isolating RNA from serum samples, continue with reverse transcription to obtain more stable complementary DNA for dPCR.
To begin, thaw the reverse transcription kit on ice. Then spin the enzymes and the primers down. Next, prepare the master mix as described in the text protocol.
Combine 10 microliters of the master mix with five microliters of extracted RNA in each well of a 96-well plate. Then centrifuge the plate at 2, 000xg at four degrees Celsius for two minutes. Proceed with the reverse transcription as detailed in the text protocol.
First, thaw the commercial dPCR mix, cDNA and PCR primers on ice and centrifuge immediately before use. Then prepare the master mix as described in the text protocol. Add 1.33 microliter volumes of reverse transcription product to 18.67 microliters volumes of the master mix and centrifuge briefly.
Using a pipet, transfer 20 microliters of the sample into each well of the medium row of an eight well cassette. Then pipet 70 microliters of droplet generation oil into the oil wells of the cassette. Next, place a gasket on top of the cassette and place it in the droplet generator.
Close the droplet generator and wait until the three indicator lights are solid green. Using a pipet, carefully transfer 40 microliters of the droplet-formed sample into separate wells of a 96-well plate. Then seal the plate with dPCR foil at 180 degrees Celsius for four seconds.
Place the sealed PCR plate in the cycler and follow the cycling instructions outlined in the text protocol. Remove the plate from the thermocycler and transfer it to the base of a plate holder. Then place the top of the plate holder onto the PCR plate.
Finally, start the commercial dPCR software. In this protocol, digital PCR was used to quantify microRNAs in the serum of patients with cardiovascular disease. Samples were taken from 16 patients before, eight hours after, and 16 hours after percutaneous intervention.
Patients with ST-elevation myocardial infarction showed a significant increase in miR-499 levels compared to patients with stable coronary artery disease. Once mastered, this technique can be done in about an hour for droplet formation of 96 samples if it is done correctly. While attempting this procedure, it is important to keep in mind to handle the droplets with care.
After watching this video, you should have a good understanding of how to perform digital PCR correctly, by correct droplet formation, cycling, reading, and analyzing.
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Overview
This study presents a protocol for the extraction and quantification of circulating microRNAs as potential biomarkers for cardiovascular diseases. By utilizing digital PCR, the method aims to enhance the accuracy of miRNA measurement in serum samples from patients with cardiovascular conditions.
Key Study Components
Area of Science
- Cardiovascular disease
- Biomarkers
- MicroRNA analysis
Background
- MicroRNAs are small non-coding RNAs involved in gene regulation.
- They have emerged as potential biomarkers for various diseases, including cardiovascular conditions.
- Digital PCR offers advantages over traditional methods for quantifying microRNAs.
- Understanding miRNA profiles can aid in the diagnosis and management of cardiovascular diseases.
Purpose of Study
- To validate microRNAs as biomarkers for cardiovascular disease.
- To demonstrate the effectiveness of digital PCR in quantifying circulating miRNAs.
- To provide a reliable protocol for researchers in the field.
Methods Used
- Extraction of miRNAs from serum samples.
- Reverse transcription to synthesize complementary DNA.
- Digital PCR for absolute quantification of miRNAs.
- Comparison of digital PCR with quantitative real-time PCR.
Main Results
- Digital PCR demonstrated superior accuracy in miRNA quantification.
- The method allows for the analysis of thousands of droplets without the need for standard curves.
- Potential applicability of the method extends beyond cardiovascular diseases to other conditions like cancer.
- Challenges in handling fragile droplets were noted for new users of the technique.
Conclusions
- The study supports the use of circulating microRNAs as biomarkers for cardiovascular diseases.
- Digital PCR is a promising tool for precise miRNA quantification.
- Further research could expand the application of this method to other diseases.
Frequently Asked Questions
Executive Industry Relevance
Quantifying circulating microRNAs with digital PCR provides a reproducible, high-sensitivity approach for biomarker validation in cardiovascular disease research. This method supports target validation and assay development by enabling absolute quantification without standard curves, reducing variability in multi-center studies. The technique enhances predictive confidence in early discovery by linking miRNA expression to pathology, informing go/no-go decisions in therapeutic hypothesis interrogation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying disease-specific miRNAs like miR-499 in AMI.
- Operational Value: Provides absolute miRNA copy numbers without standard curves, improving reproducibility across sites.
- Predictive Value: Supports biomarker-led portfolio triage by linking miRNA levels to cardiovascular pathology.
Screening & Assay Development
- Scientific Value: Generates validated biological systems (cDNA from serum RNA) for downstream screening workflows.
- Operational Value: Delivers high linearity and low variability outputs suitable for assay standardization.
- Scalability: Enables processing of 96 samples in approximately one hour post-mastered, supporting medium-throughput screening.
Translational & Preclinical Research
- Translational Continuity: Connects discovery-phase miRNA quantification to preclinical validation via disease-relevant serum biomarkers.
- Mechanistic De-risking: Reduces ambiguity in target validation by correlating miRNA expression with cardiovascular injury.
- Biomarker Alignment: Supports translational biomarker development by quantifying miRNAs with high tissue and disease specificity.
Pipeline & Workflow Integration
The method fits within the discovery continuum from hypothesis testing to lead identification, providing quantitative miRNA data that informs target confidence and assay readiness.
- Discovery Biology: Supports hypothesis testing by quantifying miRNA release from cardiovascular cells into circulation.
- Screening: Produces assay-ready, standardized outputs with high sensitivity for evaluating biomarker candidates.
- Analytics: Enables direct comparison of miRNA levels across conditions using absolute quantification without standard curves.
- Translational Research: Advances biomarker continuity by linking circulating miRNA profiles to cardiovascular pathology.
- Enterprise Reuse: Establishes a reusable quantification platform applicable across cardiovascular and oncological indications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in biomarker validity through low-variability, high-sensitivity miRNA quantification.
- Operational Value: Ensures reproducibility and standardization via droplet-based partitioning eliminating duplicate measurements.
- Strategic Value: Improves go/no-go decisions by reducing mechanistic ambiguity in biomarker-target relationships.
- Portfolio Impact: Enables risk-adjusted advancement through quantitative, pathology-linked miRNA data.
Implementation Considerations
- Requires expertise in RNA isolation, reverse transcription, and droplet handling to prevent sample loss.
- Needs droplet generation equipment, PCR cyclers, and dPCR software for accurate quantification.
- Demands cross-team standardization of droplet formation and sealing protocols for reproducible results.
- Involves adaptation considerations when applying to different sample types beyond serum, such as plasma or tissue lysates.
- Includes practical limitations like fragile droplet handling, which may affect throughput for inexperienced users.
