Preparation of breast milk samples
6 mother-newborn pairs were classified into BMJ group if newborns were clinically diagnosed with BMJ and another 6 pairs of healthy newborns were allocated into control group. None of the recruited newborns had the UGT1A1 mutation. A volume of 10 mL of human milk were collected from each mother. The milk samples were centrifuged at 2,000 × g for 15 min at 4 °C to remove cells and fat globules. Then, the supernatants were centrifuged at 12,000 × g for 30 min and further filtered through a 0.22-um PVDF filter to eliminate cells and cellular debris. Finally, the supernatants were stored at −80 °C until exosome enrichment and RNA extraction.
Preparation of exosome
Approximately 4 mL of the supernatant was next utilized for exosome preparation, using an exoRNeasy Serum/Plasma Maxi Kit (QIAGEN GmbH; Hilden, Germany)) as specified in the manufacturer’s protocol.
Transmission electron microscopy (TEM)
The prepared exosome samples were placed in contact with a support membrane of copper mesh grids for 3–5 min. The grids were then air-dried for a few minutes. Further, the grids were stained with 3% phosphotungstic acid and dried for several minutes under a filament lamp. Finally, a transmission electron microscope JEM1230 (JEOL, Tokyo, Japan) was used to visualize the grids and take photos.
Nanoparticle tracking technology (NTA)
After dilution in PBS, the exosome size and concentration were measured by nanoparticle tracking analysis (NTA) using ZETASIZER Nano series-Nano-ZS (Malvern Instruments Ltd., Malvern, UK) based on the Stokes-Einstein principle.
Western blot analysis
Twenty micrograms of protein were separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (GE Healthcare). The membranes were then blocked and incubated overnight with either a mouse anti-CD63 (1:1,000, Abcam) or rabbit anti-calnexin antibody (1:1,000, Novus). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG was used as a secondary antibody (diluted 1:2,000 in PBST). The bands obtained were scanned using ImageQuant LAS 4,000 mini densitometer (GE Healthcare Life Sciences).
Flow cytometry (FCM)
For the FCM analysis, 30 mg of exosomes (or 30 mg of FCS proteins as negative control) were incubated with 10 uL of 4-um diameter aldehyde/sulfate latex beads (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at room temperature in a final volume of 30–100 uL, followed by 2 h of gentle shaking in 1 mL PBS. The reaction was terminated by incubation for 30 min in 100 mM glycine. Exosome- or FCS-coated beads were next washed three times in FAM wash (3% FCS and 0.1% NaN3 in PBS) and resuspended in 500 uL of FCM wash. A volume of 10 uL of coated beads was incubated for 1 h with PE-conjugated CD63 Ab and then washed and analyzed using a FAM Calibur.
Enrichment of exosomal RNA from the breast milk
The stored milk supernatants (3 mL) were filtered through a 0.22-um PVDF filter to eliminate cells and cellular debris. Approximately 2 mL of the supernatant was used for total RNA exosome preparation with the exoRNeasy Serum/Plasma Maxi Kit (QIAGEN) following the manufacturer’s instructions.
Small RNA sequencing
To isolate small RNA from the prepared total RNA, we separated RNA segments of different sizes by PAGE gel, selected the 18–30-nt size range, and recycled. Then, we linked a 5-adenylated, 3-blocked single-stranded DNA adapter to the 3' end of the selected small RNAs, and the RT primer was added and crossed to the RNA 3' and excessive free 3' adapter. 5' adaptor was then also linked to 5' end of the product. Finally, reverse extension of the RT primer was performed to synthesize strand cDNA. High-ping polymerase was utilized to amplify cDNA by PCR amplification. The PCR product with a size of 100–120 bp was separated by PAGE gel to eliminate the primer dimers and other byproducts. Further, the BGISEQ-500 sequencer (BGI-Shenzhen, Shenzhen, China) was employed to obtain raw data of small RNA. Raw sequencing data were subjected to data cleaning analysis first, which included elimination of the low-quality tags, 5' adaptor contaminants, from the 50-nt tags, and obtain credible clean tags. Then, the length distribution of the clean tags and common and specific sequences in the samples were established. The standard analysis will annotate the clean tags into different categories and those which cannot be annotated to any category are used to predict the novel miRNA. After obtaining the miRNA results, target prediction for miRNAs and GO enrichment and KEGG pathway for target genes will be analyzed.
Prediction of differential expressed miRNA target genes.
The computational prediction of miRNA targets is a critical initial step in the identification of miRNA: mRNA target interactions for experimental validation. Several software was used to find the possible targets in this research. The intersection targets were identified using appropriate filter conditions such as MFE, and the score was used for further analysis. In this study, the authors used miRanda and TargetScan to predict target.
Realtime PCR of miRNAs whose predicted target genes concluded UGT1A1
The milk of another 20 mother-newborn pairs, including BMJ group (n=10) and control group (n=10), was used for quantification of differentially expressed miRNAs of which the predicted target genes concluded UGT1A1. This study used the method of realtime PCR of pre-amplified miRNA (Table3-6 in supplement). Reverse transcription was performed using the miScript PCR Starter Kit (Qiagen Hilden, Germany), and then the reverse transcribed cDNA was diluted 5-fold. The pre-amplification system was prepared using a miScript PreAMP PCR Kit (Qiagen Hilden, Germany). The pre-amplified cDNA was diluted 20-fold with RNase-free water, gently mixed and placed on ice. After preparing a real-time PCR system, add 9 μl of the above mixture and 1 μl of cDNA to the quantitative tube, then cover the tube, mix gently, centrifuge briefly and prepared for realtime PCR.
The target miRNA and the external reference of each sample were simultaneously subjected to Realtime PCR reaction, and the data were analyzed by 2-ΔΔ Ct method.