MiRNA sequencing in raw and processed human breast milk

We report the application of miRNA next generation sequencing (NGS) for the analysis of impact of processing on miRNA in human breast milk, donated by 3 volunteers. MiRNA content of total and exosomal fraction was compared between unprocessed milk and sample subjected to either Holder (thermal) pasteurization (HoP) or elevated pressure processing (HPP). NGS reads were mapped to miRBase in order to obtain miRNA counts. Then, we analyzed differences in the miRNA abundance and function between raw and processed material. It was observed that both processing methods reduce number of miRNA reads and HoP is significantly more detrimental to miRNA than HPP. Mature breast milk in a volume of 150 ml was obtained from 3 donors at the 50th day of lactation. Each sample was immediately aliquoted into 3 equal volumes for further processing. 3 samples were subjected to HPP in 62.5°C for 30 minutes in Human Milk Pasteurizer S90 Eco (Sterifeed, Medicare Colgate Ltd, England, Cullompton), 3 to HoP using U 4000/65 apparatus (Unipress Equipment, Poland, Celestynow) and 3 were left untreated as control. Then, the samples were stored frozen at -20°C. Exosomes were isolated from 5 ml of milk using miRCURY Exosome Cell/Urine/CSF Kit (Qiagen, Hilden, Germany). MiRNA was isolated with a biofluid-tailored Serum/Plasma Advanced Kit (Qiagen). The cDNA sequencing libraries were prepared with use of QIAseq miRNA Library Kit (Qiagen), according to manufacturer’s protocol. Single-end sequencing was performed on NextSeq 500 sequencer (Illumina, USA, San Diego), according to standard manufacturer’s protocol.