Sybr green hs mix

Total RNA was isolated using the Bio-Rad Aurum RNA mini-isolation kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized by the Mint reverse transcriptase kit and oligodT primer (Evrogen). After that, qPCR was performed with ready to use SYBR Green HS mix (Evrogen) and the primers specific to the EGFR, PDGFRA, TNFA, BDNF, VEGFA, ITGA2, ITGA3, ITGV, HSP60, and KLF4 genes (Supplementary Table S1); coding EGFR; platelet-derived growth factor receptor alpha (PDGFRα); tumor necrosis factor-alpha (TNF-α); brain-derived neurotrophic factor (BDNF); vascular endothelial growth factor A (VEGFA); integrin subunits alpha 2, 3, 5, and V; heat shock protein 60 (HSP60); and Krüppel-like factor 4 (KLF4). Negative controls contained all the components of the PCR mixture with cDNA replaced by mRNA gave no signal. PCR reactions were carried out using the Roche LightCycler 96 amplifier (Roche, Basel Switzerland). The mRNA expression level was normalized to the S18 ribosomal RNA level for EVs composition analysis and to the S18 and RPL13a genes expression to study the EVs’ influence on the KLF4 expression using the LightCycler SW software (Roche).

Total RNA was isolated with the Bio-Rad Aurum RNA isolation kit (Bio-Rad, Hercules, CA, USA) according to manufacturer instructions. cDNA was synthesized by the Mint reverse transcriptase kit (Evrogen, Moscow, Russia). After that, qPCR was performed with ready to use SYBR Green HS mix (Evrogen) and primers specific to the ACCN2, ACCN1, ACCN3, ACCN4, SCNN1A, and SCNN1G genes (Table 1) using the Roche LightCycler 96 amplifier (Roche, Basel, Switzerland).
Data were analyzed by the ∆∆Ct method and LightCycler SW software (Roche), and the gene expression was normalized to the expression of β-ACTIN, GPDH, and RPL13a housekeeping genes.

Total RNA was isolated using the Bio-Rad Aurum RNA isolation kit (Bio-Rad) according to the manufacturer’s instructions. cDNA was synthesized by the Mint reverse transcriptase kit (Evrogen). After that, qPCR was performed with the ready-to-use SYBR Green HS mix (Evrogen) and primers specific to the ACCN2 (coding the ASIC1a isoform, PubMed Gene ID NM_020039.4), ACCN1, ACCN3, ACCN4, SCNN1A, and SCNN1G genes (Table S2) using the Roche LightCycler 96 amplifier (Roche, Basel Switzerland). Data were analyzed by the ΔΔCt method and LightCycler SW software (Roche), and the gene expression was normalized to the expression of β-ACTIN, GPDH, and RPL13a housekeeping genes.

Total RNA from the homogenate of the cerebellum was isolated using the Bio-Rad Aurum RNA mini-isolation kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from 2 µg of total RNA by the Mint reverse transcriptase kit and oligodT primer (Evrogen, Moscow, Russia). After that, qPCR was performed with ready-to-use SYBR Green HS mix (Evrogen), 200 ng of cDNA, and the primers specific to the mouse Lynx1, Lynx2, Lypd6, Lypd6b, Psca, Slurp1, Slurp2, Chrna7, Tnfa, and Klf4 genes (Table S1); these coded Lynx1, Lynx2, Lypd6, Lypd6b, prostate-specific cell antigen PSCA, SLURP-1, SLURP-2, α7-nAChR, Tnfα, and KLF4 proteins, respecively. Negative controls containing all the components of the PCR mixture with cDNA replaced by mRNA gave no signal. PCR reactions were carried out using the Roche LightCycler 96 amplifier (Roche, Basel, Switzerland). The mRNA expression level was normalized to the β-actin, Gpdh, and RPL13a housekeeping genes using the LightCycler SW 1.0 software (Roche).