one step reverse transcription method  (Bio-Rad)

Journal: Journal of Virology

Article Title: Suppression of Host Innate Immune Response by Hepatitis C Virus via Induction of Autophagic Degradation of TRAF6

doi: 10.1128/JVI.01365-16

Figure Lengend Snippet: Suppression of TRAF6 expression by HCV. Huh7 cells were infected with HCV at a multiplicity of infection (MOI) of 1. At 3 h postinfection (hpi), the inoculum was removed, and cells were incubated in fresh medium. Cells were harvested at the time points indicated for immunoblot analysis (A) or for real-time RT-PCR analysis for TRAF6 mRNA (B) or HCV RNA (C). In panel B, the TRAF6 mRNA level in mock-infected cells was arbitrarily set at 1. The values shown in panels B and C are the means plus standard errors of the means (SEM) (error bars) from at least three independent experiments.

Article Snippet: Real-time quantitative PCR was conducted using the SYBR green-based one-step reverse transcription-PCR (RT-PCR) method (catalog no. 4392653; Applied Biosciences).

Techniques: Expressing, Infection, Incubation, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Suppression of Host Innate Immune Response by Hepatitis C Virus via Induction of Autophagic Degradation of TRAF6

doi: 10.1128/JVI.01365-16

Figure Lengend Snippet: Effects of TRAF6 on HCV replication. (A) Huh7 cells were transfected with the control vector or the plasmid that expressed the Flag-tagged TRAF6 (FLAG-TRAF6) for 24 h and then mock infected or infected with HCV for 24 h or 48 h. Cell lysates were then collected for immunoblot analysis. (B) Huh7 cells infected by HCV as mentioned above for panel A were lysed for quantification of HCV RNA using real-time RT-PCR. The HCV RNA levels were normalized against GAPDH RNA. (C to E) Huh7 cells were transfected twice with the control siRNA or the TRAF6 siRNA and then infected with HCV for 24 or 48 h with an MOI of 1. The cells were then lysed for immunoblot analysis (C) or real-time RT-PCR analysis for quantification of HCV RNA (D and E). The incubation medium was also harvested at 24 and 48 h for titration of infectious HCV particles using the focus formation assay as previously described (46). Values that are significantly different are indicated by asterisks as follows: *, P < 0.05; **, P < 0.005.

Article Snippet: Real-time quantitative PCR was conducted using the SYBR green-based one-step reverse transcription-PCR (RT-PCR) method (catalog no. 4392653; Applied Biosciences).

Techniques: Transfection, Plasmid Preparation, Infection, Western Blot, Quantitative RT-PCR, Incubation, Titration, Tube Formation Assay

Journal: Journal of Virology

Article Title: Suppression of Host Innate Immune Response by Hepatitis C Virus via Induction of Autophagic Degradation of TRAF6

doi: 10.1128/JVI.01365-16

Figure Lengend Snippet: Effects of TRAF6 on the NF-κB promoter and the expression of cytokines in HCV-infected cells. (A) Huh7 cells were transfected with the NF-κB-luciferase reporter and either the control siRNA or the TRAF6 siRNA for 24 h. The CMV-renilla luciferase reporter was also used for cotransfection to monitor transfection efficiencies. The cells were then infected with HCV for 24 h or 48 h and then lysed for measuring the luciferase activities using the Dual-Luciferase assay kit (Promega). The results represent the means plus SEM from three independent experiments. Huh7 cells transfected with the control siRNA or the TRAF6 siRNA were infected with HCV for 24 or 48 h. (B) Cells were then lysed for quantification of IL-6 or TNF-α (TNFa) mRNAs using real-time RT-PCR. The levels of IL-6 and TNF-α mRNAs of mock-infected cells transfected with the control siRNA were arbitrarily set at 1. (C) The incubation medium was collected and filtered at 24 and 48 h postinfection for analysis of IL-6 and TNF-α by ELISA.

Article Snippet: Real-time quantitative PCR was conducted using the SYBR green-based one-step reverse transcription-PCR (RT-PCR) method (catalog no. 4392653; Applied Biosciences).

Techniques: Expressing, Infection, Transfection, Luciferase, Cotransfection, Quantitative RT-PCR, Incubation, Enzyme-linked Immunosorbent Assay