Droplet digital polymerase chain reaction-based quantification of circulating microRNAs using small RNA concentration normalization.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
02 06 2020
02 06 2020
Historique:
received:
20
12
2019
accepted:
12
05
2020
entrez:
4
6
2020
pubmed:
4
6
2020
medline:
18
12
2020
Statut:
epublish
Résumé
Quantification of plasma microRNAs (miRNAs) as non-invasive disease biomarkers is subject to multiple technical variabilities. This study aimed to develop an optimized protocol for miRNA quantification from rodent plasma. We hypothesized that a fixed small RNA concentration input for reverse transcription (RT) reaction will provide better miRNA quantification than a fixed RNA volume input. For this, tail-vein plasma was collected from 30 naïve, adult male Sprague-Dawley rats. Plasma hemolysis was measured with NanoDrop-1000 and Denovix DS-11 spectrophotometers. Plasma was then pooled, and RNA was extracted from 50-μl, 100-μl or 200-μl pool aliquots. Small RNA concentration was measured with Qubit miRNA assay. A fixed RNA volume (un-normalized) or a fixed small RNA concentration was used for RT (concentration-normalized). The method was setup with miR-23a-3p and validated with miR-103a-3p and miR-451a. Hemolysis measurements from Denovix and NanoDrop strongly correlated. Qubit revealed increased small RNA concentrations with increasing starting plasma volumes. With concentration-normalization, miRNA levels from 100-µl and 200-µl plasma volume groups mostly normalized to the level of the 50-µl in ddPCR. Our results indicate that miRNA quantification with ddPCR should be performed with small RNA concentration-normalization to minimize variations in eluted RNA concentrations occuring during RNA extraction.
Identifiants
pubmed: 32488181
doi: 10.1038/s41598-020-66072-z
pii: 10.1038/s41598-020-66072-z
pmc: PMC7265372
doi:
Substances chimiques
Circulating MicroRNA
0
MIRN103 microRNA, rat
0
MIRN23 microRNA, rat
0
MIRN451A microRNA, rat
0
MicroRNAs
0
Edetic Acid
9G34HU7RV0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
9012Références
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