Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Binderup HG

PROVIDER: S-EPMC6053236 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Binderup Helle Glud HG Madsen Jonna Skov JS Heegaard Niels Henrik Helweg NHH Houlind Kim K Andersen Rikke Fredslund RF Brasen Claus Lohman CL

PloS one 20180719 7

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 dua ...[more]

PMID: 30024941

Similar Datasets

Project description:The relationship between the expression of microRNAs (miRNAs) in blood and a variety of diseases has been investigated. MiRNA-based liquid biopsy has attracted much attention, and cancer-specific miRNAs have been reported. However, the results of analyses of the expression of these miRNAs vary among studies. The reproduction of results regarding miRNA expression levels could be difficult if there are differences in the data acquisition process. Previous studies have shown that the anticoagulant type used during plasma preparation and sample storage conditions could contribute to differences in measured miRNA levels. Thus, the impact of these preanalytical conditions on comprehensive miRNA expression profiles was examined. First, the miRNA expression profiles of samples obtained from healthy volunteers were analyzed using next-generation sequencing. Based on an analysis of the library concentration, human genome identification rate, ratio of unique sequences and expression profiles, the optimal preanalytical conditions for obtaining highly reproducible miRNA expression profiles were established. The optimal preanalytical conditions were as follows: ethylenediaminetetraacetic acid (EDTA) as the anticoagulant, whole-blood storage at room temperature within 6 hours, and plasma storage at 4°C or -20°C within 30 days. Next, plasma samples were collected from 60 cancer patients (3 facilities × 20 patients/facility), and miRNA expression profiles were analyzed. There were no significant differences in measurements except in the expression of erythrocyte-derived hsa-miR-451a. However, the variation in hsa-miR-451a levels was smaller among facilities than among individuals. This finding suggests that samples obtained from the same facility could show significantly different degrees of hemolysis across individuals. We found that the standardization of anticoagulant use and storage conditions contributed to reducing the variation in sample quality across facilities. The findings from this study could be useful in developing protocols for collecting samples from multiple facilities for cancer screening tests.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets