Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

Oriol Timoneda,Sarai Córdoba,Ingrid Balcells,Armand Sánchez,Anna Castelló,Atsushi Asakura

doi:10.1371/journal.pone.0044413

Oriol Timoneda, Sarai Córdoba + Show 4 more

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https://doi.org/10.1371/journal.pone.0044413

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Abstract

Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r2 = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.

Highlights

MicroRNAs are small non-coding RNAs involved in gene expression regulation at the post-transcriptional level in animals, plants and viruses [1,2,3]
Analysis of the Stability of the Reference miRNAs In accordance with the most stable miRNAs described in the literature [20,21,22,24,26,27,28], ten candidate miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, SscmiR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and SscmiR-17-5p, Table 1) were selected to study their expression stability in different porcine tissues and breeds
All candidate reference miRNAs were successfully amplified through Reverse transcription (RT)-qPCR, allowing us to perform adequate genetic expression quantification [32]

Summary

Introduction

MicroRNAs (miRNAs) are small non-coding RNAs involved in gene expression regulation at the post-transcriptional level in animals, plants and viruses [1,2,3]. They participate in a wide range of biological processes where they play important roles. MiRNA expression has been associated with different pathological processes, such as cancer, neurological disorders, inflammatory pathologies and cardiovascular diseases [5,6,7,8]. It is very important to measure the miRNA expression with high accuracy

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