Quantification of microRNA in plasma using probe based TaqMan assays: is microRNA purification required?

OBJECTIVE: Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. RESULTS: We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.

Identification of aspirin resistance using a PDW-miR92a-score: Validation in an intermittent claudication cohort.

OBJECTIVE: Aspirin is a widely used platelet inhibitor to prevent thrombotic events. However, in 25% of patients the antiplatelet effect is insufficient. The current study aimed to validate a newly developed PDW-miR92a-score as a biomarker of the individual response to aspirin enabling targeted antithrombotic therapy. METHODS: Blood samples were collected from 209 patients with intermittent claudication on daily aspirin therapy. Based on results from the arachidonic acid stimulated aggregation test, patients were defined as aspirin resistant (n = 92) or responders (n = 117). Using the cut-off values for platelet distribution width (PDW) and plasma levels of microRNA-92a (miR-92a) defined in our pilot study, we investigated the performance of the combined PDW-miR92a-score in the validation study. Furthermore, receiver operating characteristic curve analysis was performed in the validation cohort in order to optimize the cut-off values of the two score parameters. RESULTS: PDW and miR-92a levels were significantly higher in aspirin resistant compared to responding patients. When using the predefined cut-off values for PDW and miR-92a the combined PDW-miR92a score showed high specificity (93.1%) but poor sensitivity (19.8%) for aspirin resistance. By recalculation using new cut-off values identified in the validation cohort, a score with a specificity of 75% and a sensitivity of 54.9% was obtained. CONCLUSION: Both PDW and plasma levels of miR-92a were confirmed to be significantly higher in aspirin resistant compared to responding patients in our validation cohort. We were, however, unable to confirm the high sensitivity of the combined PDW-miR92a-score previously published by our group in a pilot study.

Quantification of microRNA levels in plasma - Impact of preanalytical and analytical conditions.

Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2-6.8% and 10.5-31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4-20.1%, and the inter-assay variation 5.7-26.7%. Plasma preparation and microRNA purification were found to account for 39-73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

Aspirin resistance may be identified by miR-92a in plasma combined with platelet distribution width.

OBJECTIVE: Aspirin is a widely used drug for prevention of thrombotic events in cardiovascular patients, but approximately 25% of patients experience insufficient platelet inhibition due to aspirin, and remain in risk of cardiovascular events. This study aimed to investigate the value of circulating miR-92a and platelet size as biomarkers of the individual response to aspirin therapy. METHODS: Blood samples were collected from 50 healthy blood donors without antithrombotic medication and 50 patients with intermittent claudication on daily aspirin therapy. Based on results from the arachidonic acid stimulated aggregation test on Multiplate®analyzer (ASPItest), patients were defined as aspirin resistant (n=10) or aspirin responders (n=40). Plasma levels of miR-92a were evaluated by RT-qPCR analysis and platelet distribution width (PDW) was used to assess platelet size variability. Receiver operating characteristic curves for miR-92a levels and PDW were used to set cut-off values for discrimination between aspirin responding and aspirin resistant patients. RESULTS: When defining aspirin resistance as an ASPItest ≥30U, the optimal cut-off values for discrimination of aspirin responders and aspirin resistant patients were found to be PDW>11.8fL and a relative expression level of miR-92a>4.5. Using these cut-off values we could define a PDW/miR-92a-score with a specificity of 97.5% and a sensitivity of 80.0% in relation to detect aspirin resistance. The corresponding positive and negative predictive values were found to be 88.9% and 95.1%, respectively. CONCLUSION: Aspirin resistance can potentially be identified by miR-92a levels in plasma combined with PDW.

Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples.

BACKGROUND: In the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content in platelets this may partly be explained by residual platelets in the plasma samples used. When collecting fresh plasma samples, it is possible to produce cell-free/platelet-poor plasma by centrifugation. In this study, we systematically investigated whether biobanked EDTA plasma samples could be processed to be suitable for miRNA analysis. MATERIALS AND METHODS: Blood samples were collected from ten healthy volunteers and centrifuged to produce platelet-poor-plasma (PPP) and standard biobank plasma. After one week at -80 °C the biobanked EDTA plasma was re-centrifuged by different steps to remove residual platelets. Using RT-qPCR the levels of 14 miRNAs in the different plasma preparations were compared to that of PPP. RESULTS: We were able to remove residual platelets from biobanked EDTA plasma by re-centrifugation of the thawed samples. Nevertheless, for most of the investigated miRNAs, the miRNA level was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0-1×109/L. CONCLUSION: We found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even for the miRNAs found to be less effected, we showed that a 1.5-3 fold change in plasma levels may possible be caused by or easily overseen due to sample preparation and/or storage.

A functional CD86 polymorphism associated with asthma and related allergic disorders.

BACKGROUND: Several studies have documented a substantial genetic component in the aetiology of allergic diseases and a number of atopy susceptibility loci have been suggested. One of these loci is 3q21, at which linkage to multiple atopy phenotypes has been reported. This region harbours the CD86 gene encoding the costimulatory B7.2 protein. The costimulatory system, consisting of receptor proteins, cytokines and associated factors, activates T cells and regulates the immune response upon allergen challenge. METHODS: We sequenced the CD86 gene in patients with atopy from 10 families that showed evidence of linkage to 3q21. Identified polymorphisms were analysed in a subsequent family-based association study of two independent Danish samples, respectively comprising 135 and 100 trios of children with atopy and their parents. Functional analysis of the costimulatory effect on cytokine production was performed in an autologous cell-based system based on cells expressing CD86 variants. RESULTS: Two polymorphisms were identified, encoding the amino acid changes Ile179Val and Ala304Thr, respectively. Significant associations were observed between the Ile179Val polymorphism and allergy phenotypes in both samples (eg, asthma, p = 4 x 10(-3) in the two samples combined). The undertransmitted (protective) Val179 allele was found to induce higher production of both Th1 and Th2 cytokines than the overtransmitted (risk) Ile179 allele, suggesting a functional impact of the polymorphism. CONCLUSION: The CD86 gene, and specifically the Ile179Val polymorphism, may be a novel aetiological factor in the development of asthma and related allergic disorders.

Atopic dermatitis -- a total genome-scan for susceptibility genes.

Atopic dermatitis is one of the most common chronic diseases of childhood and closely related to other clinical manifestations of allergy. The incidence is high and still increasing. The genetic contribution to disease development is substantial and complex. Only recently genetic research has begun to focus on this phenotype, and specific susceptibility genes remain to be found. To identify candidate regions holding genes for atopic dermatitis we performed a genome-scan in Danish affected sib-pair families containing sib-pairs matching a phenotype definition of both clinical atopic dermatitis and confirmed specific allergy. The scan was undertaken using 446 microsatellite markers and non-parametric linkage results were obtained from the MAPMAKER/SIBS computer program. We found evidence of linkage to three candidate regions in chromosomes 3p (MLS=2.14), 4p (MLS=2.00) and 18q (MLS=2.25), one of which has not been reported previously. Eight additional regions showed weaker but positive results.