Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction

Md Matiur Rahman; Ryoka Nakanishi; Fumi Tsukada; Shigeo Takashima; Yoshiko Wakihara; Yuji O Kamatari; Kaori Shimizu; Ayaka Okada; Yasuo Inoshima

doi:10.3390/membranes13020185

Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction

Membranes (Basel). 2023 Feb 2;13(2):185. doi: 10.3390/membranes13020185.

Authors

Md Matiur Rahman^{1

2}, Ryoka Nakanishi¹, Fumi Tsukada¹, Shigeo Takashima^{3

4

5}, Yoshiko Wakihara³, Yuji O Kamatari^{4

5

6}, Kaori Shimizu¹, Ayaka Okada^{1

7}, Yasuo Inoshima^{1

7

8}

Affiliations

¹ Laboratory of Food and Environmental Hygiene, Gifu University, Gifu 501-1193, Japan.
² Department of Medicine, Faculty of Veterinary, Animal and Biomedical Sciences, Sylhet Agricultural University, Sylhet 3100, Bangladesh.
³ Division of Genomics Research, Life Science Research Center, Gifu University, Gifu 501-1193, Japan.
⁴ Institute for Glyco-Core Research (iGCORE), Gifu University, Gifu 501-1193, Japan.
⁵ The United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan.
⁶ Division of Instrumental Analysis, Life Science Research Center, Gifu University, Gifu 501-1193, Japan.
⁷ Education and Research Center for Food Animal Health, Gifu University (GeFAH), Gifu 501-1193, Japan.
⁸ Joint Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan.

Abstract

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via qPCR analysis. Five miRNAs, bta-miR-29a, bta-miR-200a, bta-miR-26b, hsa-miR-27b-3p, and hsa-miR-30b-5p, were selected by microarray analyses, and their cycle threshold (Ct) values were further evaluated mathematically using geNorm, NormFinder, BestKeeper, and ∆Ct algorithms. The results revealed that both the miRNeasy Micro Kit and Maxwell RSC miRNA Tissue Kit are useful for the efficient recovery of RNA from bovine milk sEVs. According to the final stability ranking analyzed by RefFinder, hsa-miR-27b-3p and bta-miR-29a can be used as suitable internal control miRNAs in bovine milk sEVs. The study also indicated that using a suitable internal control miRNA may improve the reliability and accuracy of the qPCR analysis for normalization in bovine milk sEVs. To the best of our knowledge, this is the first study to uncover the suitable internal control miRNAs in bovine milk sEVs.

Keywords: RNA extraction kit; bovine milk small extracellular vesicles; internal control microRNA; normalization in quantitative polymerase chain reaction.

Abstract

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