Rapid Communication: MiR-92a as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk1

Abstract

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Bovine milk transcriptome analysis reveals microRNAs and RNU2 involved in mastitis

“…Liquid biopsy is a noninvasive method of diagnosing diseases such as cancers and infectious diseases by analyzing certain dysregulated miRNAs in the animal’s body fluids. In our previous studies, we have shown that miR‐92a is suitable for use as a housekeeping gene in the analysis of bovine mastitis‐related miRNAs in milk and that the miRNAs in milk can be used as liquid biopsy biomarkers for bovine mastitis . In the present study, we comprehensively profiled genome‐wide miRNA expression in milk from cows with bovine mastitis.…”

“…qPCR was performed as previously described . Equal volumes of RNA (1.25 μL) were reverse‐transcribed to cDNA with TaqMan miRNA Assays (Thermo Fisher Scientific), according to the manufacturer’s protocol.…”

Milk MicroRNAs in Health and Disease

“…Milk microRNAs are of particular importance because of their potential use as diagnostic, prognostic and functional tools in disease assessment and management (Alsaweed, Lai, Hartmann, Geddes, & Kakulas, ; Cui et al., ; Decmann et al., ; Iannaccone et al., ; Lai et al., , ; Markkandan et al., ; Melnik & Schmitz, ; Perge, Nagy, Decmann, Igaz, & Igaz, ; Schanzenbach, Kirchner, Ulbrich, & Pfaffl, ; Tome‐Carneiro et al., ). It is also of interest to study their impact in infant development in the context of infant formulas, which are poor in microRNAs (Alsaweed et al., ; Chen et al., ; Oh, Park, Son, & Kim, ), for newborns not fed with maternal milk (Oh et al., ; Zempleni et al., 2016).…”

“…In milk, microRNA expression was related to mammary gland conditions, including infections (Iannaccone et al., ; Lai et al., , ; Sun et al., ), cancer (Decmann et al., ; Do Canto et al., ; Hesari et al., ), pregnancy and lactation status (Carney et al., ; Perri et al., ; Schanzenbach et al., ), and maternal diet (Chen et al., ). Therefore microRNAs in milk were studied for their potential as biomarkers (Gustafson, Tyryshkin, & Renwick, ; Melnik & Schmitz, ) such as quality controls of commercial milk (Oh et al., ).…”

“…Some of the first links between milk microRNA and disease or development were explored in bovine milk in view of using milk microRNAs as biomarkers for mammary gland infections; especially mastitis (Lai et al., , ;), staphylococcus infection (Cai et al., ; Sun et al., ), and tuberculosis (Iannaccone et al., ). In these studies, immunity‐related microRNAs were overexpressed in milk samples from infected cows, thereby allowing their use as biomarkers of disease.…”