Dataset Information

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

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.

SUBMITTER: Rahman MM

PROVIDER: S-EPMC9961204 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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

Rahman Md Matiur MM Nakanishi Ryoka R Tsukada Fumi F Takashima Shigeo S Wakihara Yoshiko Y Kamatari Yuji O YO Shimizu Kaori K Okada Ayaka A Inoshima Yasuo Y

Membranes 20230202 2

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 the ...[more]

PMID: 36837688

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Project description:ObjectiveQuantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs.MethodsThe cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins.ResultsBased on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance.ConclusionThis study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

Dataset Information

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

Publications

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

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