Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 2;15(4):e0231040.
doi: 10.1371/journal.pone.0231040. eCollection 2020.

MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health

Lene B Dypås  1 , Kristine B Gützkow  1 , Ann-Karin Olsen  1 , Nur Duale  1
Affiliations

MiRNA profiles in blood plasma from mother-child duos in human biobanks and the implication of sample quality: Circulating miRNAs as potential early markers of child health

Lene B Dypås et al. PLoS One. .

Abstract

Background: MicroRNAs (miRNAs) have been linked to several diseases and to regulation of almost every biological process. This together with their stability while freely circulating in blood suggests that they could serve as minimal-invasive biomarkers for a wide range of diseases. Successful miRNA-based biomarker discovery in plasma is dependent on controlling sources of preanalytical variation, such as cellular contamination and hemolysis, as they can be major causes of altered miRNA expression levels. Analysis of plasma quality is therefore a crucial step for the best output when searching for novel miRNA biomarkers.

Methods: Plasma quality was assessed by three different methods in samples from mother-child duos (maternal and cord blood, N = 2x38), with collection and storage methods comparable to large cohort study biobanks. Total RNA was isolated and the expression profiles of 201 miRNAs was obtained by qPCR to identify differentially expressed miRNAs in cord and maternal plasma samples.

Results: All three methods for quality assurance indicate that the plasma samples used in this study are of high quality with very low levels of contamination, suitable for analysis of circulating miRNAs. We identified 19 significantly differentially expressed miRNAs between cord and maternal plasma samples (paired t-tests, FDR<0.05, and fold change>±1.5), and we observed low correlation of miRNA transcript levels between cord and maternal samples throughout our dataset.

Conclusions: Our findings suggest that good quality plasma samples suitable for miRNA profiling can be achieved from samples collected and stored by large biobanks. Incorporation of extensive quality control measures, such as those established here, would be beneficial for future projects. The overall low correlation of miRNA expression between cord and maternal samples is an interesting observation, and promising for our future studies on identification of miRNA-based biomarkers in cord blood plasma, considering that these samples were collected at term and some exchange of blood components between cord and maternal blood frequently occur.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The presence of residual platelets and microparticles in plasma samples.
The presence of residual platelets and microparticles in (A) plasma samples from cord (n = 38) and (B) maternal blood (n = 38) was measured using a CASY Cell Counter and Analyzer Model TT with LOD = 0.7. Results are presented as counts/μl by particle diameter (μm).
Fig 2
Fig 2. Spectrophotometric analysis of plasma to assess the degree of hemolysis from oxyhemoglobin absorbance at 414 nm.
Absorbance in plasma was measured for (A) cord blood (n = 38), (B) maternal blood (n = 38), and a hemolyzed reference sample (in red). Average absorbance at 414 nm for the plasma samples from cord and maternal blood was 0.18 ±0.015 (range: 0.05–0.44) and 0.25 ±0.020 (range: 0.03–0.48), respectively. Absorbance at 414 nm for the hemolyzed reference sample was 1.12.
Fig 3
Fig 3. Distribution of ΔCq = Cqhsa-miR-23a - Cqhsa-miR-451 in plasma samples as a measure of hemolysis.
The transcript level of hsa-miR-23a was compared with the transcript level of hsa-miR-451a to assess the degree of hemolysis in plasma samples from cord blood (n = 38), maternal blood (n = 38), and a hemolyzed reference sample. Average ΔCq for the plasma samples from cord and maternal blood was 2.78 ±0.17 and 2.50 ±0.19, respectively. The hemolyzed reference sample had a ΔCq of 12.13.
Fig 4
Fig 4. Correlation of expression levels in plasma from cord and maternal blood.
Linear regression of log2-transformed NRQ-values for maternal plasma samples as a function of cord samples to assess correlation of expression levels (A) before and (B) after removal of potential outliers.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008;18(10):997–1006. 10.1038/cr.2008.282 - DOI - PubMed
    1. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105(30):10513–8. 10.1073/pnas.0804549105 - DOI - PMC - PubMed
    1. Esteller M. Non-coding RNAs in human disease. Nat Rev Genet. 2011;12(12):861–74. 10.1038/nrg3074 - DOI - PubMed
    1. Fiore R, Schratt G. Releasing a tiny molecular brake may improve memory. EMBO J. 2011;30(20):4116–8. 10.1038/emboj.2011.356 - DOI - PMC - PubMed
    1. Kloosterman WP, Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell. 2006;11(4):441–50. 10.1016/j.devcel.2006.09.009 - DOI - PubMed

Publication types

Grants and funding

This work was supported by The Research Council of Norway, NFR-FREE MEDBIO grant, POEMA - POTENTIAL EARLY DIAGNOSTIC MOLECULAR MARKERS OF ADHD: Analysis of miRNA profiles and DNA methylation status in triads (grant no.: 240763/F20). We would also like to thank the European Union Integrated Project NewGeneris, 6th Framework Programme, Priority 5: Food Quality and Safety; Newborns and Genotoxic exposure risks (FOOD-CT-2005-016320). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.