Rnalater ice solution

Around 50–75 mg of whole human lung tissues were pre-treated with RNAlater-ICE Solution (Invitrogen, Life Technologies, Carlsbad, CA, USA, AM7030) and processed with a Polytron tissue homogenizer. RNA was then extracted according to the TRIzol protocol (TRIzol, Life Technologies, Carlsbad, CA, USA). Using the Nanodrop 2000c (Thermo Scientific), RNA concentrations were determined spectrophotometrically, and all samples underwent RNA quality control (RIN > 6). To generate the first-strand cDNA, the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR was used according to the manufacturer’s instructions (Invitrogen by Life Technologies, Carlsbad, CA, USA, #11752). The quantitative PCR was run in triplicates using the TaqMan Universal Master Mix II (Applied Biosystems, Thermo Scientific, Vilnius, Lithuania, #4440040). TaqMan primers for AGER (Life Technologies, Carlsbad, CA, USA, Cat#Hs00542584_g1) and GAPDH (housekeeper; Life Technologies, Carlsbad, CA, USA, Cat#Hs02758991_g1) were used.

Total RNA was extracted from HCC cell lines HepG2 and Huh7 and immortalized normal liver PH5CH cells using Qiagen miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA was also extracted from two Grade-2 HCC tumor tissues (HCC103T, HCC105T, BioChemed) and a normal liver tissue using mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). All kits were used by following manufacturer guidelines. Tumor samples corresponding to two plasma samples (HCC103P, HCC105P, BioChemed) were investigated by RNAseq and one of them (HCC103T) was also studied by RT-qPCR. 100 mg tissue treated with RNAlater-ICE solution (Invitrogen, Carlsbad, CA, USA) was homogenized in a glass homogenizer, worked-up following kit protocol, and RNA eluted in 100 μl RNase-free water. Plasma (3 ml) from HCC101P (BioChemed) was spun at 2,000g for 15 minutes to remove cellular debris and supernatant was collected. Total RNA from extracellular vesicles (EVs) was extracted using the ExoMir Kit (BIOO Scientific Corp., Austin, TX, USA). RNA from the remaining EV-free flow-through plasma was extracted using the Qiagen RNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). RNA from plasma EVs, EV-free plasma, and liver tissues was sequenced at TJU as described above.