Exosomes were separated from tubal fluid by gradient centrifugation and ultracentrifugation as MISEV reported (Witwer et al., 2021)(Théry et al., 2018). We used nanoparticle tracking analysis (NTA) ZetaVIEW S/N 17-310 (Particle Meerbusch, Germany) to measure the diameter of exosomes (Supplementary Fig.1A). Exosomes were also dyed by uranyl acetate and detected by TEM (Supplementary Fig.1B&C) (Di Pietro, 2016).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and purified using mirVana™ miRNA Isolation Kit without phenol (Cat # AM1561, Ambion, Austin, TX, US), following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit (Cat # 5190-0456, Agilent technologies, Santa Clara, CA, US) followed the manufacturer’s instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit (Cat # 5190-0456, Agilent technologies, Santa Clara, CA, US) in hybridization Oven (Cat # G2545A, Agilent technologies, Santa Clara, CA, US) at 55°C，20 rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After hybridization, slides were washed in staining dishes (Cat # 121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat #5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat # G2565CA, Agilent technologies, Santa Clara, CA, US) and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) with default settings. Raw data were normalized by Quantile algorithm, included in the R package AgiMicroRna(López-Romero, P. BMC Genomics. 2011.).
Description
Exosomal miRNA microarray intubal fluid
Data processing
Putative target genes of miRNAs were predicted by miRanda. R package ClusterProfiler was used to perform enrichment analysis of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Pathways were also enriched by DAVID (https://david.ncifcrf.gov). KEGG analysis was performed simultaneously using the ClusterProfiler package of R software and the DAVID website to predict the pathways.
Exosomal microRNAs in tubal fluid may be involved in tubal epithelial structure damage and reproductive dysfunction associated with tubal endometriosis.
