Itaq universal probes mastermix

Tissue samples were lysed in QIAzol Lysis reagent (Qiagen, Maryland, USA), homogenized with stainless steel beads in TissueLyser II (Qiagen, Maryland, USA) and incubated on ice for five minutes followed by miRNA isolation using Direct-zol RNA MiniPrep kit with on column DNase digestion (Zymo Research, Irvin, CA). Reverse transcription (30 min −16 °C; 30 min −42 °C; 5 min −85 °C) was performed in Eppendorf Mastercycler (Eppendorf, New York, USA) using 10 ng RNA, TaqMan primers and MiRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), followed by quantitative RT-PCR (10 min −95 °C; 40 cycles of 15 sec −95 °C; 1 min −60 °C) in CFX96 (Bio-Rad Laboratories, Hercules, CA) using TaqMan Universal PCR Master Mix (Biorad, Hercules, CA). All tissue results were normalized to snoRNA202 expression. Serum sample controls were spiked with Caenorhabditis elegans (cel)-miR-39, as per instructions (Qiagen, Gaithersburg, MD) and subsequently analyzed utilizing a primer pool. Briefly, serum cDNA synthesis was performed with a final 0.1x primer concentration and Applied Biosystems’s rtPCR kit followed by individual target-specific RT-qPCR analysis using Bio-Rad iTaq Universal Probes Master Mix according to manufacturer instructions.

RNA was extracted from cells with the Qiagen RNeasy protocol (Catalog No. 74004). Quality control and quantification were performed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA was aliquoted and stored at −80°C. cDNA synthesis was performed using high-capacity cDNA Reverse Transcription Kit from Thermo Fisher Scientific (4368814) and iTaq/universal probes mastermix (Bio-Rad, 1725131) was used for single-gene RT-qPCR reactions (Supplementary Table S2). Chemokine expression was quantified using RT² Profiler PCR array for mouse chemokines/cytokines (Qiagen PAMM-150ZA, 330231) with data analysis performed using the Qiagen online tool PCR Array data analysis Web portal (https://www.qiagen.com/gb/resources/resourcedetail?id=20762fd2-8d75-4dbe-9f90-0b1bf8a7746b&lang=en). The chemokines tested, quality control, and normalization analysis are found in Supplementary Tables S3 to S5.