Quickgene 810

The experimental methods were approved by the Hankyong National University Animal Ethics Committee, Anseong, Republic of Korea (No.2021-1). A total of 269 whole blood samples were obtained from the four populations of the five geographically distinct provinces (Table 1, Figure 1). Sampling sites included i) East Mongolia Khentii Province (KTP, n = 53); ii) West Mongolia Uvs Province (USP, n = 43); iii) Southern Mongolia Omnogovi and Dundgovi provinces (GOP, n = 133); and iv) Northern Mongolia Khovsgol province (KGP, n = 40). Genomic DNA was extracted from blood samples using the methods described for QuickGene 810 (Kurabo, Osaka, Japan). The concentration and purity of the extracted genomic DNA were evaluated using an ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Cells were treated with GGA or other compounds, and total RNA was isolated using the QuickGene RNA cultured Cell kit S (Wako) with QuickGene-810 (Kurabo, Osaka, Japan). cDNA was generated using the Transcriptor First Strand cDNA Synthesis kit with random hexamers (Roche Diagnostics, Basel, Switzerland). Nucleotide sequences of the PCR primers for the DDIT3 (DNA- damage-inducible transcript 3, also known as CHOP), PDIA4 (protein disulfide isomerase family A, member 4), ACSL3 (acyl-CoA synthetase long-chain family member 3) and 28S rRNA cDNAs are listed in S1 Table in S1 File. Real-time PCR was performed with DyNAmo Capillary SYBR Green qPCR Master mix (Finnzymes, Espoo, Finland), and cDNA on LightCycler1.5 (Roche Diagnostics) under conditions described in S2-S7 Tables in S1 File.

For mRNA quantification, RNA was extracted from cultured cells by the QuickGene RNA cultured cell kit S (RC-S) and a 50 μL volume of RNA was obtained using QuickGene-810 equipment, according to the manufacturer’s instructions (KURABO, Osaka, Japan). In a 20 μL reaction, total RNA (0.5 μg) was converted to cDNA using the reverse transcription Takara PrimeScript RT Master Mix (TaKaRa Bio, Shiga, Japan) and diluted using qPCR Takara EASY Dilution. The Brilliant III SYBR® MM with ROX PCR master mix (Agilent Technologies, CA) was used to prepare the diluted cDNA samples for quantification by the Agilent Mx3000P QPCR system. The following primer sets were used for mRNA quantification: TARDBP, 5′-CCCCAGATATTGCCAATATC-3′ and 5′-AAGTTTCCAATATGCTCAATTAAG-3′; Precore, 5′-GGTCTGTTCACCAGCACCAT-3′ and 5′-GGAAAGAAGTCAGAAGGCAA-3′; Core, 5′-CCGGAAAGCTTGAGCTCTTCTT-3′ and 5′-CACAGAATAGCTTGCCTGAGTG-3′; APOA2 5′-CAACTGTGCTACTCCTCACCAT-3′ and 5′-TGGAAGTACTGAGAAACCAGG C-3′; Total HBV RNA, 5′-GAGTGCTGTATGGTGAGGTG-3′ and 5′-TTTGGGGCATGGACATTGAC-3′. Quantification of HBV mRNAs (precore and pregenomic) was performed as we described recently^{62 (link)}. Gene expression was normalized to that of GAPDH. Relative mRNA levels represent the level of the gene divided by the level of GAPDH mRNA.