Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions
Cell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data no...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-02-01
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| Series: | Animals |
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| Online Access: | https://www.mdpi.com/2076-2615/13/4/576 |
| _version_ | 1797622873569886208 |
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| author | Gabriella Guelfi Camilla Capaccia Michele Matteo Santoro Silvana Diverio |
| author_facet | Gabriella Guelfi Camilla Capaccia Michele Matteo Santoro Silvana Diverio |
| author_sort | Gabriella Guelfi |
| collection | DOAJ |
| description | Cell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data normalization allows an accurate cross-sample gene expression comparison. The appropriate selection of EC is a crucial step because qPCR data can change drastically when normalization is performed using an unstable versus a stable EC. To find EC cmiRNA with stable expression in search and rescue (SAR) working dogs, we explored the serum miRNome by Next-Generation Sequencing (NGS) at T0 (resting state) and T1 immediately after SAR performance (state of physiologically recovered stress). The cmiRNAs selected in the NGS circulating miRNome as probable ECs were validated by qPCR, and miRNA stability was evaluated using the Delta Ct, BestKeeper, NormFinder, and GeNorm algorithms. Finally, RefFinder was used to rank the stability orders at both T0 and T1 by establishing miR-320 and miR-191 as the best-circulating ECs. We are confident that this study not only provides a helpful result in itself but also an experimental design for selecting the best endogenous controls to normalize gene expression for genes beyond circulating miRNAs. |
| first_indexed | 2024-03-11T09:16:25Z |
| format | Article |
| id | doaj.art-dfb84f27d7d142bba082cd42c278051f |
| institution | Directory Open Access Journal |
| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-11T09:16:25Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj.art-dfb84f27d7d142bba082cd42c278051f2023-11-16T18:38:31ZengMDPI AGAnimals2076-26152023-02-0113457610.3390/ani13040576Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress ConditionsGabriella Guelfi0Camilla Capaccia1Michele Matteo Santoro2Silvana Diverio3Laboratory of Ethology and Animal Welfare (LEBA), Department of Veterinary Medicine, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia, ItalyDepartment of Veterinary Medicine, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia, ItalyGdF (Military Force of Guardia di Finanza), Dog Breeding and Training Course, Via Lungolago 46, 06061 Castiglione del Lago, ItalyLaboratory of Ethology and Animal Welfare (LEBA), Department of Veterinary Medicine, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia, ItalyCell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data normalization allows an accurate cross-sample gene expression comparison. The appropriate selection of EC is a crucial step because qPCR data can change drastically when normalization is performed using an unstable versus a stable EC. To find EC cmiRNA with stable expression in search and rescue (SAR) working dogs, we explored the serum miRNome by Next-Generation Sequencing (NGS) at T0 (resting state) and T1 immediately after SAR performance (state of physiologically recovered stress). The cmiRNAs selected in the NGS circulating miRNome as probable ECs were validated by qPCR, and miRNA stability was evaluated using the Delta Ct, BestKeeper, NormFinder, and GeNorm algorithms. Finally, RefFinder was used to rank the stability orders at both T0 and T1 by establishing miR-320 and miR-191 as the best-circulating ECs. We are confident that this study not only provides a helpful result in itself but also an experimental design for selecting the best endogenous controls to normalize gene expression for genes beyond circulating miRNAs.https://www.mdpi.com/2076-2615/13/4/576endogenous controlqPCRcirculating miRNAsearch and rescue dogsphysiological stress |
| spellingShingle | Gabriella Guelfi Camilla Capaccia Michele Matteo Santoro Silvana Diverio Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions Animals endogenous control qPCR circulating miRNA search and rescue dogs physiological stress |
| title | Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions |
| title_full | Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions |
| title_fullStr | Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions |
| title_full_unstemmed | Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions |
| title_short | Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions |
| title_sort | identification of appropriate endogenous controls for circulating mirna quantification in working dogs under physiological stress conditions |
| topic | endogenous control qPCR circulating miRNA search and rescue dogs physiological stress |
| url | https://www.mdpi.com/2076-2615/13/4/576 |
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