Exorneasy serum plasma maxi isolation kit

Total RNA was isolated from the serum exosomes using the exoRNeasy Serum/Plasma Maxi Isolation Kit (Qiagen, Germany) in accordance with the manufacturer’s instructions. RNA was re-suspended in 14 μL RNase-free water and used immediately or frozen at −80°C. Small-RNA cDNA libraries were generated using NEBNext Multiplex Small RNA Library Prep Set for Illumina^® (NEB, United States) following the manufacturer’s recommendations. The library preparations were sequenced on an Illumina Novaseq 6000 platform, and 50 bp single-end reads were generated. The small RNA tags were mapped to the reference sequence by Bowtie (Langmead et al., 2009 (link)) without mismatch to analyze their expression and distribution on the reference. The miRNA expression levels were estimated by transcript per million (Zhou et al., 2010 (link)). Differential expression analysis of two conditions was performed using the DEGseq (2010) R package. P-values were converted to adjusted P-values using the Benjamini–Hochberg method. The adjusted P < 0.05 and | log2 (fold change)| > 1 were set as the thresholds for significantly differential expression. All the procedures for small-RNA library preparation and sequencing analysis were accomplished by Novogene (Beijing, China).

Total RNA was isolated from the serum exosomes using the exoRNeasy Serum/Plasma Maxi Isolation Kit (Qiagen, Germany) according to the manufacturer’s protocol. Isolated RNAs were validated by RT-qPCR on the enlarged serum samples (n = 8 per group). cDNA was generated using the TransScript^® miRNA First-Strand cDNA Synthesis Super Mix (TransGen, China) and diluted for use in 10 μL qPCR reactions using Thermo SYBR Green kits in a QuantStudio 7 Flex Real-Time PCR System (life, United States). Thermocycler settings were: 95°C for 5 min, followed by 40 cycles of 95°C for 20 s, 60°C for 15 s, and 72°C for 20 s, followed by 3 min at 72°C. The primers used in the validation assays were designed on the basis of miRNA mature sequence and listed in Supplementary Table S1. The exogenously added spike-in cel-miR-39 (Qiagen, Germany) was used as the reference (Salvoza et al., 2016 (link)). The relative expression levels were calculated using the 2^–ΔΔCt method and analyzed using SPSS version 21.0 (SPSS, Inc., Chicago, IL, United States). Paired t-tests and two-way analyses of variance were performed for analysis. P < 0.05 indicated a statistically significant difference.