Circulating microRNA biomarkers for cardiovascular risk prediction: are we approaching clinical application?

Ronde, Maurice W J de; Pinto, Yigal M.; Pinto‐Sietsma, Sara‐Joan

doi:10.21037/atm.2016.12.05

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…For example, a microRNA that is detected in all samples of group A but not detected in any sample of group B, is importantly differentially regulated between groups A and B. By excluding all missing values, this significant difference would be lost [ 22 ]. A better approach is to replace Cq values >35 or undetermined with an arbitrary low value, as we did in our approach [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…By excluding all missing values, this significant difference would be lost [ 22 ]. A better approach is to replace Cq values >35 or undetermined with an arbitrary low value, as we did in our approach [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…Raw Cq values were calculated in SDS software v.2.4 using automatic baseline and threshold settings. Cq values that were undetermined or >35 were replaced by “Cq = 36” for further analysis to minimize statistical confounding by high quantification cycle values [ 22 ]. Relative miRNA levels were expressed as 2 -ΔCq .…”

Section: Methodsmentioning

confidence: 99%

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MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation

et al. 2018

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MicroRNA (miRNA) regulate gene expression through posttranscriptional mRNA degradation or suppression of translation. Many (pre)analytical issues remain to be resolved for miRNA screening with TaqMan Low Density Arrays (TLDA) in plasma samples, such as optimal RNA isolation, preamplification and data normalization. We optimized the TLDA protocol using three RNA isolation protocols and preamplification dilutions. By using 100μL elution volume during RNA isolation and adding a preamplification step without dilution, 49% of wells were amplified. Informative target miRNA were defined as having quantification cycle values ≤35 in at least 20% of samples and low technical variability (CV across 2 duplicates of 1 sample <4%). A total of 218 miRNA was considered informative (= 59% of all target miRNA). Different normalization strategies were compared: exogenous Ath-miR-159a, endogenous RNA U6, and three mathematical normalization techniques: geNorm (Qbase, QB) and NormFinder (NF) normalization algorithms, and global mean calculation. To select the best normalization method, technical variability, biological variability, stability, and the extent to which the normalization method reduces data dispersion were calculated. The geNorm normalization algorithm reduced data dispersion to the greatest extent, while endogenous RNA U6 performed worst. In conclusion, for miRNA profiling in plasma samples using TLDA cards we recommend: 1. Implementing a preamplification step in the TLDA protocol without diluting the final preamplification product 2. A stepwise approach to exclude non-informative miRNA based on quality control parameters 3. Against using snoRNA U6 as normalization method for relative quantification 4. Using the geNorm algorithm as normalization method for relative quantification.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation

et al. 2018

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“…Many pre-analyti-cal and analytical parameters remain incompletely defined and may affect the quantification of circulating miRNAs as well as the interpretation of data. These parameters refer to the overall analysis process, from the first step that includes the biological sample type (serum vs. plasma) to the collection and isolation of miRNA protocols, and methods for miRNA detection, normalization, and possible application of the pre-amplification procedures (4)(5)(6)(7)(8). Pre-amplification is a method that allows for the analysis of genes with a limited amount of nucleic acids by increasing the number of copies of nucleotide sequences in the reaction prior to polymerase chain reaction (PCR) analysis while maintaining target amplification specificity (8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs

Sekovanić

Dorotić²,

Jurasović

et al. 2021

Biochem. med. (Online)

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Introduction The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. Materials and methods Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. Results Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (C T ) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). Conclusion Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification.

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Elevated miRNA-499 Levels in Early Phase of Non-ST Elevation Acute Coronary Syndromes Predict Increased Long-Term Risk of Major Adverse Cardiac Events

Miśkowiec,

Szymczyk,

Wejner-Mik

et al. 2024

JCM

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Background/Objectives: Available data suggest the diagnostic potential of testing microRNAs (miRs) in myocardial infarction, but their prognostic value remains unclear. To evaluate the prognostic value of circulating miRs (miR-1, miR-21, miR-133a, miR-208 and miR-499) for predicting major adverse cardiac events (MACEs), including death, non-fatal myocardial infarction (MI) or cardiovascular rehospitalization, in patients with non-ST segment elevation acute coronary syndromes (NSTE-ACS). Methods: Our prospective, single-center, observational study included patients (pts) with NSTE-ACS admitted <24 h after symptoms onset and pts with confirmed stable coronary artery disease (SCAD) as controls. Relative expression of miRs was calculated, and subjects were categorized according to miRs expression on hospital admission into two groups (≤median and >median). Results: Overall, 103 NSTE-ACS (52 NSTEMI/51 UA) and 47 SCAD pts (median age 66 years, 67% male) were included. During the median 895 (581–1134) days of the follow-up, MACE occurred in 75 (50%) patients: 20 (13%) died, 28 (19%) presented with MI, and 65 (43%) were readmitted due to cardiovascular reasons. Incidence of MI, rehospitalization and MACE was significantly higher in pts with elevated (>median) miR-499 [MI: 34.3% vs. 7.3%; HR = 6.0 (2.8–12.7) for rehospitalization; 53.7% vs. 36.2%, HR = 2.3 (1.4–3.8) for MACE; 62.7% vs. 42%, HR = 2.4 (1.5–3.8)] for hospital readmission. In the Cox proportional hazards regression model, miR-499 expression above the median level [HR = 1.8 (1.1–3.1)], high-sensitivity cardiac troponin T [HR = 1.2 (1.02–1.5)], diabetes [HR = 1.7 (1.1–2.8)] and percutaneous intervention during hospital stay [HR = 2.1 (1.1–3.8)] were identified as independent predictors of MACE in long-term observation, even after adjustment for covariates. Conclusions: Elevated miR-499 level on hospital admission in NSTE-ACS is related to an increased rate of MACE in the 2.5-year follow-up.

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Circulating microRNA biomarkers for cardiovascular risk prediction: are we approaching clinical application?

Cited by 3 publications

References 12 publications

MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation

MicroRNA profiling in plasma samples using qPCR arrays: Recommendations for correct analysis and interpretation

Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs

Elevated miRNA-499 Levels in Early Phase of Non-ST Elevation Acute Coronary Syndromes Predict Increased Long-Term Risk of Major Adverse Cardiac Events

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