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MicroRNA from Dried Blood Spots: a Novel Biomarker Method in Newborns after Perinatal Asphyxia

Presented at the Neonatal Society 2015 Summer Meeting (programme).

Ponnusamy V1,2, Kapellou O3, Yip E1, Evanson J4, Michael-Titus A5, Yip P5, Shah D1,2

1 Centre of Paediatrics, Blizard Institute, UK
2 NICU, The Royal London Hospital, UK
3 NICU, Homerton Hospitals, UK
4 Imaging Services, The Royal London Hospital, UK
5 Centre of Neuroscience, Blizard Institute, UK

Background: MicroRNAs (miRNAs) are non-coding RNAs involved in regulating gene expression. Their study from dried blood spot (DBS) in newborns and their role as a biomarker for hypoxic-ischemic encephalopathy (HIE) for therapeutic hypothermia (TH) are yet to be explored. Hypotheses:1) Extraction and quantification of candidate miRNAs from DBS correlate with miRNA levels obtained from biofluids including plasma, blood-EDTA and urine. 2) Expression of neuro-specific miRNAs correlate with MRI outcomes in babies after TH.

Methods: Ethically approved newborns with HIE were prospectively recruited; Group 1 fulfilled standard criteria and received TH and Group 2, with mild HIE did not. Blood, a DBS and urine were collected; plasma was separated from the blood-EDTA. RNA extraction and quantitative RT-PCR were performed on all samples to extract and quantify three candidate miRNAs (RNU6B, Let7b and miR-21). MRIs were reported independently by 2 reviewers.

Results: Of 30 neonates recruited, 19 received TH. 13/19 were predicted to have a favourable outcome based on MRI of cerebral tissue injury described by Rutherford et al (1). Overall, 80% (24/30) were predicted to have a favourable neurological outcome. MiRNAs were extractable from all samples of both groups. Prolonged storage of DBS (range 2 to 191 days) at room temperature did not affect the expression for all 3 miRNAs in plasma, blood and DBS indicating stability of miRNAs. There was a significant positive correlation between DBS and EDTA-blood for RNU6B (R2=0.27, p=0.005), let7b (R2=0.50, p=0.0001) and miR-21 (R2=0.20, p=0.01). The comparison of miRNA expression using mean Ct values for all three miRNAs analysed demonstrated significantly lower Ct values, meaning higher expression in DBS than in plasma, EDTA-blood and urine in both the predicted unfavourable as well as favourable outcome groups of newborns.

Conclusion: It is feasible to study the expression of miRNA from dried blood spots obtained from newborns. This novel method using a very small drop of blood will be valuable for future research of miRNAs as biomarkers for brain injury and the potential for development of other objective bedside biomarkers of brain injury in the newborn.

Corresponding author:

1. Rutherford M, et al. Assessment of brain tissue injury after moderate hypothermia in neonates with hypoxicischemic encephalopathy: a nested substudy of a randomised controlled trial. Lancet Neurol 2010;9:39-45.

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