Iscript reverse transcription system kit

10–20 mg of liver tissue was homogenized in QIAzol lysis reagent (QIAGEN, Valencia, CA, USA) using stainless steel beads via TissueLyser II (QIAGEN, Valencia, CA, USA). Total RNA was extracted using the miRNeasy kit (QIAGEN, Valencia, CA, USA) as recommended by the manufacturer.⁶⁰ (link) For mRNA analysis, cDNA was transcribed with the iScript reverse transcription system kit (Bio-Rad, Hercules, CA, USA), and quantitative real-time PCR was performed via CFX96 iCycler (Bio-Rad, Hercules, CA, USA). Quantitative analyses of genes were performed using gene-specific primers as presented in Table S2 and as described previously.⁶⁵ (link) Cq value was normalized to 18S or β actin mRNA, and differential expression fold changes were calculated using the delta-delta Ct method. For miR analysis, TaqMan miR assays (Applied Biosystems, Foster City, CA, USA) were used as described earlier.¹⁴ (link)^,60 (link) SnoRNA-202 (mouse samples) or RNU48 (human samples) were used to normalize the technical variations between the samples.

10–20 mg of each organ was homogenized in QIAzol Lysis reagent (Qiagen, Germany) using stainless steel beads in TissueLyser II (Qiagen, USA). Total RNA was isolated using Direct-zol RNA MiniPrep kit with on column DNA digestion (Zymo Research Corp., USA). For mRNA analysis, cDNA synthesis was carried out using iScript reverse transcription system kit (BioRad, USA) and quantitative analyses of genes were performed using gene-specific primers on a Bio-Rad iCycler real time machine. Primer sequences for TNFα, MCP1, IL-1β and 18S were same as described28 (link). Cq value was normalized to 18S and fold change was calculated using the delta-delta Ct method. For the detection of miRNAs, TaqMan miRNA Assays (Applied Biosystems) were employed as described previously28 (link). SnoRNA202 (tissue/cells) or synthetic cel-miR-39 (exosomes and plasma) was used to normalize the technical variations between the samples. To enhance miR-155 signal in recipient miR-155 KO mice after the administration of WT plasma (miR-155 enriched) a pre-amplification step was introduced. The cDNA was pre-amplified using pre-amplification kit as described by the supplier (Applied Biosystem, USA) and diluted pre-amplified product was used for real time PCR.