Journal of Blood & Lymph

Selection and Optimization of Circulating Exosomal Reference microRNAs for Quantitative Real-time PCR Studies of Porcine Body Fluids

Abstract

Qiang Pu, Lin Wang, Guojun Kang, Changbao Liu, Changfeng Yang, Jia Luo* and Yongfu Huang*

Circulating exosomal miRNAs released into all body fluids have incredible functionality and stability. Their expression is associated with multiple pathological conditions, can be used as informative biomarkers when assessing and monitoring the body’s physiopathological status. However, there is no consensus on reference miRNAs for circulating exosomal reference and abundance normalization. The present study aimed to quantify 16 potential reference miRNAs in ten porcine body fluids using qRT-PCR. Further, their stability was quantified by combining multiple gold-standard statistical tools, including BestKeeper, GeNorm, and NormFinder. The identified miRNAs were comprehensively ranked. The top-ranked miRNA was recommended as the optimal reference miRNAs for data normalization. To identify more stable genes, the body fluids were assigned into three groups based on the collection point, they are in vivo (bile, bladder fluid, and gastric juice), in vitro (colostrum, ordinary milk, semen, and urine) and in the blood (UVBP, UABP and PBS). The most stable optimal circulating exosomal reference miRNAs in the body fluids were let-7b-5p (miR-93) in bile, miR-92a in bladder fluid, miR-93 in gastric juice, let-7b-5p in colostrum, miR-92a in ordinary milk and urine, miR-25 in semen, let-7b-5p in UVBP, miR-25 in UABP and U6 in PBS. Overall, miR-93, miR-451 (miR-92a), and miR-25 are the bona fide reference miRNA for qRT-PCR data normalization of body fluids in vivo, in vitro, and blood, respectively. Across all body fluids, miR-451 was the most stable when determining the miRNA abundance in the circulating exosomes