Parker, V.L., Cushen, B.F., Gavriil, E. et al. (4 more authors) (2021) Comparison and optimisation of microRNA extraction from the plasma of healthy pregnant women. Molecular Medicine Reports, 23 (4). 258. ISSN 1791-2997
Abstract
Circulating microRNA (miRNA) biomarkers are implicated in the diagnosis, monitoring and prediction of various disease processes. Before embarking upon biomarker discovery, miRNA extraction techniques must first be optimised in the biofluid and population under study. Using plasma from a healthy pregnant woman, it was attempted to optimise and compare the performance of two commercially available miRNA extraction kits; Qiagen (miRNeasy Serum/Plasma) and Promega (Maxwell® RSC miRNA from Tissue or Plasma or Serum). Sample miRNA content (concentration and percentage) was assessed using Agilent Bioanalyzer Small RNA chips and reverse transcription‑quantitative PCR (RT‑qPCR) using four constitutively expressed miRNAs (hsa‑miR‑222‑3p, hsa‑let‑7i‑3p, hsa‑miR‑148‑3p and hsa‑miR‑30e‑5p). Quality control spike‑ins monitored RNA extraction (UniSp2, 4 and 5) and cDNA synthesis (UniSp6, cel‑miR‑39‑3p) efficiency. Optimisation approaches included: i) Starting volume of plasma; the addition of ii) Proteinase K; iii) a RNA bacteriophage carrier (MS2); and iv) a glycogen carrier. The two kits exhibited equivalence in terms of miRNA recovery based on Bioanalyzer and RT‑qPCR ΔΔCq results. Optimisation attempts for both kits failed to improve upon miRNA content compared with standard methodology. Comparing the standard methodology, the Qiagen kit was more consistent (smaller variance of ΔCq values) compared with the Promega kit. The standard methodology of either kit would be suitable for the investigation of miRNA biomarkers in a healthy pregnant population.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2021 The Authors. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (https://creativecommons.org/licenses/by-nc-nd/4.0/) License. |
Keywords: | microRNA; Homo sapiens; extraction; optimisation; pregnancy; women |
Dates: |
|
Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Sheffield Teaching Hospitals |
Funding Information: | Funder Grant number Weston Park Hospital Cancer Charity CA154 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 12 Mar 2021 14:21 |
Last Modified: | 12 Mar 2021 14:21 |
Status: | Published |
Publisher: | Spandidos Publications |
Refereed: | Yes |
Identification Number: | 10.3892/mmr.2021.11897 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:171716 |