Twenty-five bags of randomly selected RBC suspensions with anticoagulants (ACD-A) were provided by the Central Blood Bank of Deyang City, Sichuan Province, China. Exosomes from five bags were used for gene chip, and the others were used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation of miRNA expressions. Meanwhile, the research protocol was approved by the ethics committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences.
Storage of RBC suspensions and Isolation and Purification of Exosome
RBC suspensions were split into 100mL aliquots in100-mL transfer bags and stored at 4°C. The aliquots of RBC samples were used for exosome isolation after storing for 1, 3, and 5 weeks. The aliquots of RBC suspensions were centrifuged at 3000 g for 20 minutes to obtain supernatants. Exosomes were isolated from the supernatants using the ultracentrifugation method, with an initial speed at 300g for 10 min, followed by sequential centrifugation at 2000g for 10 min, 10,000g for 30min to separate supernatant, then 100,000g for 70 min to separate exosome pellets. Exosome precipitation was resuspended in 200 µL of phosphate-buffered saline (PBS), and 0.22-μm centrifugal filter (Millipore, USA) was used to remove large extracellular vesicles (EVs).
Identification of Exosome
The isolated exosomes from RBC suspensions were verified by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and Western Blot analysis (WB).
The particle size distribution was measured by NTA with NanoSight NS300 instrument (Malvern Instruments, UK). The size of the particles was analyzed using DTS v5.10 software (Malvern Panalytical, UK) and the results were given as particle size distribution.
For TEM analysis, copper grids were placed in the exosome suspensions fixed with 2% paraformaldehyde overnight. The morphology of the isolated exosomes was visualized by TEM (FEI Tecnai™ G2 Spirit, Czech Republic) at 80 kV.
Exosomes were treated with RIPA lysis buffer, and protein concentration was calculated using the method of bicinchoninic acid (BCA). Absorbance values were detected by Varioskan LUX (Thermo Fischer Scientific, USA). Exosome protein markers against TSG101 (Abcam, UK), CD9 (Abcam, UK), and CD63 (Santa Cruz, USA) were validated by WB analysis. Protein marker against Calnexin was also detected as a negative control. Lysates of Hela cells were tested as control samples. Signals of the membranes were captured and imaged by ChemiScope Mini 3000 (CLINX, Shanghai).
RNA isolation and cDNA preparation
Total RNA was extracted from exosomes using QRIzol reagent and miRNeasy Mini Kit (Qiagen, German). The quantity and quality of the total extracted RNA were measured by using Agilent 4200 platform and Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA). Only high-quality RNA samples were used for subsequent experiments. The miScript II RT Kit with miScript hispec buffer (Qiagen, German) was used to prepare cDNA for subsequent gene chip and qRT-PCR validation.
Microarray analysis was performed by Gminix Biotechnology Company (Shanghai, China) using Affymetrix GeneChip miRNA 4.0 array (Affymetrix, USA). Gene chips were then scanned using the GeneChip® Scanner 3000 7G (Affymetrix). CEL-files of the raw data were exported and then uploaded to the website, Gminix-Cloud Biotechnology Information (GCBI), of Genminix Informatics Co., Ltd. (Shanghai, China; http://www.gcbi.com.cn) for further analysis. The data were analyzed through the Robust Multichip Analysis algorithm (RMA) using Affymetrix default analysis settings and global scaling as a normalization method. Then we used the successive approximation method (SAM) for differentially expressed miRNAs analysis. According to the filter condition | Fold Change | >1.2 & P value <0.05, the final differential result was obtained.
Bioinformatics analysis of differentially expressed exosomal miRNAs
The target genes of differentially expressed exosomal miRNAs were predicted by miRBase and TargetScan. The gene ontology (GO) analysis of target genes was performed using a P<0.05 to define statistically enriched GO categories. Pathway analysis was used to determine the significant pathway of the differential genes according to the Kyoto Encyclopedia of Genes and Genomes database (http://www.genome.jp/kegg/). The miRNAs–genes network was built to show the interactions of miRNAs and their target genes.
Identification of miRNA expression by qRT-PCR
According to the results of gene chip, there were 17 miRNAs upregulated more than 10 folds at 5weeks compared to 1week storage time. These miRNAs were selected and their expression changes were validated by qRT-PCR. Twenty bags of RBC suspension samples were used for validation. qRT-PCR was performed following the manufacturer’s protocol of miScript SYBR PCR kit (Qiagen, German) and using different miRNA primers for each cDNA sample of reverse transcription. The miRNA qPCR primers were synthesized by RIBOBIO Biotech (Guangzhou, China). Cel-miRNA-39 (Qiagen, German) was used to normalize potential sample-to-sample variations and technical variations. Relative expression changes of miRNAs were analyzed through the method of 2-ΔΔCT.