Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Hardikar, Anandwardhan A.; Farr, Ryan J.; Joglekar, Mugdha V.

doi:10.1161/jaha.113.000792

Cited by 54 publications

(46 citation statements)

References 14 publications

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“…Their application needs to be defined for specific indications to minimize deviations between studies. Importantly, the method of normalization needs to be standardized and reproducible isolation and detection protocols should be applied; otherwise, comparability is considerably hampered [155]. Apart from these analytical aspects, there are influencing parameters affecting miRNA levels in the analyzed biomaterial.…”

Section: Analytical Considerations In Mirna Quantificationmentioning

confidence: 99%

microRNAs in cardiovascular disease – clinical application

Schulte

Karakas

Zeller

2017

Clinical Chemistry and Laboratory Medicine (CCLM)

104

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microRNAs (miRNAs) are well-known, powerful regulators of gene expression, and their potential to serve as circulating biomarkers is widely accepted. In cardiovascular disease (CVD), numerous studies have suggested miRNAs as strong circulating biomarkers with high diagnostic as well as prognostic power. In coronary artery disease (CAD) and heart failure (HF), miRNAs have been suggested as reliable biomarkers matching up to established protein-based such as cardiac troponins (cT) or natriuretic peptides. Also, in other CVD entities, miRNAs were identified as surprisingly specific biomarkers -with great potential for clinical applicability, especially in those entities that lack specific protein-based biomarkers such as atrial fibrillation (AF) and acute pulmonary embolism (APE). In this regard, miRNA signatures, comprising a set of miRNAs, yield high sensitivity and specificity. Attempts to utilize miRNAs as therapeutic agents have led to promising results. In this article, we review the clinical applicability of circulating miRNAs in CVD. We are giving an overview of miRNAs as biomarkers in numerous CVD entities to depict the variety of their potential clinical deployment. We illustrate the function of miRNAs by means of single miRNA examples in CVD.

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Section: Analytical Considerations In Mirna Quantificationmentioning

confidence: 99%

microRNAs in cardiovascular disease – clinical application

Schulte

Karakas

Zeller

2017

Clinical Chemistry and Laboratory Medicine (CCLM)

104

View full text Add to dashboard Cite

show abstract

“…It should be noted that one of the samples, outlined in Figure 4C, displays 4-fold less U6 content than that presented in Figure 4B. Since the level of U6 in sample presented in 4C is 75% less to begin with than the one presented in panel 4B, greater technical variability is expected due to the Poisson distribution of transcripts, which is exacerbated by the small reaction volume 17 .…”

Section: Representative Resultsmentioning

confidence: 89%

“…Research has been conducted to identify abundant miRNAs in different cells or tissues, as well as in circulation from human plasma and serum samples, which is more accessible and less invasive 9,[11][12][13][14][15] . Different methods of miRNA quantification have been established using multiple platforms, such as the standard 96-well plate platform 4,12,[16][17][18] , the microfluidics card platform 12,[18][19][20][21][22][23] and the nanofluidics array platform 17,24 . Quantitative real-time PCR (qPCR) offers the ability to measure relative or absolute numbers of transcripts using multiple (dye-or probe-based) chemistries.…”

Section: Introductionmentioning

confidence: 99%

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Wong

Farr

Joglekar

et al. 2015

JoVE

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Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

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“…79,[82][83][84] As a result, often PCR cycle numbers (Cq) distributions are skewed relative to actual miRNA copy numbers, even with the latest locked nucleic acid (LNA) technology. [85][86][87][88][89][90][91][92][93] The same multiplex qRT-PCR test can have limits of detection that are different by several orders of magnitude for different virus serotypes. 94,95 The problem of discordant results between different platforms becomes worse in the case of low viral loads.…”

Section: Quantitative Reverse Transcription-pcr (Qrt-pcr)mentioning

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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Circulating microRNAs: Understanding the Limits for Quantitative Measurement by Real‐Time PCR

Cited by 54 publications

References 14 publications

microRNAs in cardiovascular disease – clinical application

microRNAs in cardiovascular disease – clinical application

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

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

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