Abstract
Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.
Highlights
MicroRNAs are small non-coding RNAs, which are widely expressed in the genomes of plants, animals, and humans [1]
The standard deviation for miR-127 and miR93 was lowest among the genes tested, indicating that they are more stably expressed than doi:10.1371/journal.pone.0122554.g001
Discussion Quantitative real-time PCR (qRT-PCR) is a popular tool for accurate miRNA expression profiling [7, 26,27,28]
Summary
MicroRNAs (miRNAs) are small non-coding RNAs, which are widely expressed in the genomes of plants, animals, and humans [1]. Reference MicroRNAs in Bovine Serum miRNAs have the potential to serve as biomarkers for changes in physiological conditions such as pregnancy and in pathological conditions such as cancer, diabetes, and other diseases. It is important to measure miRNA expression in the blood with high accuracy. Quantitative real-time PCR (qRT-PCR) is currently the most frequently used approach for the evaluation of circulating miRNAs [5,6,7]. Due to its high sensitivity, specificity, good reproducibility, and cost-effectiveness, this is a powerful technique for measuring the expression profile of miRNAs. The accuracy of qRT-PCR-based miRNA expression analysis depends on an appropriate normalization by using reference genes. The optimal selection of genes to be used for normalization is critical for qRT-PCR data analysis
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