Mispike

RT-qPCR analysis was performed to assess contamination of spin columns with small RNAs and confirm findings from sequencing analysis. RNeasy MinElute spin columns (Qiagen) might be contaminated with small RNAs, thereby causing artifacts in microRNA analysis [14 (link)]. RNeasy MinElute columns were treated with sodium hypochlorite to remove possible contamination [14 (link)]. We dissolved a non-natural microRNA (miSpike; IDT DNA, Coralville, IA; 5’-rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrU-3’) in 200 μL RNase-free, molecular biology grade water and extracted miSpike using MinElute columns. Eluted miSpike (100 ng) was reverse transcribed using the miScript II RT Kit (Qiagen). In addition, miR-30d-5p, miR-125a-5p and miR-423-5p were analyzed in hypochlorite-treated and untreated columns. RT-qPCR was performed using miScript SYBR Green (Qiagen), the universal reverse primer in the miScript II RT Kit and sequence-specific forward primers (Supplemental Table 3, Supplemental Digital Content 3). Hypochlorite-treated columns were used when confirming sequencing data by RT-qPCR. Melting curve analyses suggested formation of a single product (not shown). Ct values greater than 29 were considered not detectable [15 (link)].

RNA was purified from exosomes (from 1 mL milk) using the miRNeasye MinElute spin columns were treated with sodium hypochlorite to remove possible contamination and cDNA was synthesized by using the miScript II RT kit following the manufacturer’s recommendations (Qiagen, Germantown, MD). efficiency of RNA extraction and qPCR, a non-natural microRNA (miSpike; IDT DNA, Coralville, IA; 5’-rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrU-3’) was used in experiment as described Wang et al [16 (link)]. Quantitative real-time PCR (qPCR) was used to assess the expression of miR-30d-5p, miR-125a-5p and miR-423–5p, which are among the 10 most abundant microRNAs in human milk exosomes [11 (link)]. qPCR was conducted using the miScript SYBR green PCR kit (Qiagen) and PCR primers as previously described [11 (link)]. PCR reactions yielded a single product judged by melting curve analysis (Supplemental Figure S2) Ct values greater than 29 were considered not detectable [16 (link)].