7000 system sds software v1

Forty-eight hour after hypoxia (5 % O₂, 3 % O₂ or 1 % O₂) treatment, or 48 h after hypoxia (1 % O₂) treatment followed by As₂O₃ treatment for 24 h, total RNA fractions were isolated from HBx-HepG2 cells and HepG2.2.15 cells using TRIzol reagent (Invitrogen, CA, USA). qRT-PCR was performed with SYBR Premix Ex Taq (TaKaRa). GAPDH was used as an internal control for mRNA. The relative quantitative analysis of data was performed using 7000 system SDS software v1.2.3 (Applied Biosystems). The relative quantitation of target gene expression was obtained using the comparative ΔΔCT method.

Total RNA and miRNA fractions were isolated from blood samples, HepG2 cells and HepG2.2.15 cells using TRIzol reagent (Invitrogen, CA, USA). MiRNA extraction was performed using the miRNA Extraction Kit (Tiangen, Beijing, China). QRT-PCR was performed with SYBR Premix Ex Taq (TaKaRa). GAPDH was used as an internal control for mRNA, and RNU6B was used as the miRNA reference. The relative quantitative analysis of data were performed using 7000 system SDS software v1.2.3 (Applied Biosystems). The relative quantitation of target gene expression was obtained using the comparative ΔΔC_T method.

Reverse transcription was performed using the RevertAid first-strand cDNA synthesis kit following the manufacturer’s instructions (MBI Fermentas, Vilnius, Lithuania). RNA quantity and quality were assessed by determination of the optical density at 260 and 280 nm using spectrophotometry and additional visualization by agarose gel electrophoresis. Real-time PCR was performed in the ABI 7000 SDS (Applied Biosystems, Foster City, CA, USA) using the Power SYBR Green (Applied Biosystems) according to the manufacturer’s instructions. Briefly, cDNA was diluted 5 times and 4 μl diluted cDNA template was used for each PCR with 250 nM forward and reverse primers in a total volume of 20 μl. The thermal cycling conditions comprised 5 min at 95°C, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s. All of the reactions were performed in triplicate. The relative quantity of the target mRNA was normalized to the level of the internal control GAPDH mRNA level. The relative quantitative analyses of the data were performed using 7000 system SDS software v1.2.3 (Applied Biosystems). The relative quantitation of target gene expression was obtained using the comparative ΔΔC_T method.

Reverse transcription was performed using the RevertAid first-strand cDNA synthesis kit following the manufacturer’s instructions (MBI Fermentas, Vilnius, Lithuania). RNA quantity and quality were assessed by determination of the optical density at 260 and 280 nm using spectrophotometry and additionally by visualization in agarose gels. Real-time PCR was performed in the ABI 7000 SDS (Applied Biosystems) using the Power SYBR Green (Applied Biosystems) according to the manufacturer’s instructions. The thermal cycling conditions comprised of 5 min incubation at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s. All of the reactions were performed in triplicate. The relative quantity of the target mRNA was normalized to the level of the internal control GAPDH mRNA level. The relative quantitative analyses of the data were performed using SDS 7000 system software v1.2.3 (Applied Biosystems, USA). The relative quantitation of target gene expression was obtained using the comparative ΔΔC_T method [28 (link)].

Approximately 35 ng of total RNA was reverse-transcribed in a 10-uL volume using the TaqMan miRNA reverse-transcriptase kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Three microliters of the reverse-transcription reaction was used in each of the real-time PCR assays. Analyses of miR-122 were carried out in triplicates by means of the TaqMan human miRNA assays (Applied Biosystems) using SDS 7000 system software v1.2.3 (Applied Biosystems, USA).