Total RNA was extracted from different tissues (0.1 g liver or tissues indicated in Supplementary information, Figure S1D ), cells (1 × 106) or serum (0.1 ml) using Trizol or Trizol LS reagent (Invitrogen). Quantitative RT-PCR analyses with One-step QuantiTect SYBR Green Kit (Qiagen, CA, USA) of the indicated genes were performed as previously described14 (link) in an ABI 7500 (ABI, CA, USA).The internal standard HCV RNA was synthesized by in vitro transcription (MEGAscript T7, Ambion, TX, USA) from pJFH-1 plasmid and quantified by a commercial kit (path-HCV, PrimerDesign, UK) at 1.1 × 1010 copies/ml. Serial dilutions of the internal standard HCV RNAs were spiked in ICR sera and liver homogenates to determine the linear range of detection and the limit of detection, using One-step QuantiTect SYBR Green Kit. The limit of detection of HCV RNA by this method is defined as the lowest concentration at which 95% of HCV RNA are detected53 (link), i.e., 500 copies/ml (serum) and 100 copies/mg (liver), respectively. Nested PCR was performed to detect HCV negative strand16 (link) (see Supplementary information, Table S6 for primers). Quantitative RT-PCR analyses were carried out similarly for mRNA levels of IFNα4, IFNβ1, ISGs, miR-122, apoE, CypA and IL-28 (see Supplementary information, Table S6 for primers). Data were analyzed with ABI software version 2.0.3 (Applied Biosystems).
One step quantitect sybr green kit
Manufactured by Qiagen
Sourced in United States, Switzerland
The One-step QuantiTect SYBR Green Kit is a real-time PCR assay kit that enables the detection and quantification of RNA targets. It provides all necessary components for reverse transcription and real-time PCR amplification in a single reaction, using SYBR Green for fluorescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Megascript t7, by Thermo Fisher Scientific (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Abi software version 2, by Thermo Fisher Scientific (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Promega (1 mentions)
Trisure reagent, by Meridian Bioscience (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
2 protocols using one step quantitect sybr green kit
1
Quantitative RT-PCR Analysis of Viral and Host Transcripts
2
Antioxidant and Pro-Apoptotic Gene Expression
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The expression analysis of the antioxidant and pro-apoptotic genes was determined by RT-qPCR using a LightCycler 480 System (Roche, Basel, Switzerland) and the One-Step QuantiTect SYBR-Green Kit (Qiagen, Limburg, Netherlands). Total cellular RNA was extracted by using TRIsure reagent (Bioline, London, UK), treated with RNAse-Free DNase (Promega, Wisconsin, USA), and used as template for mRNA amplification with specific human oligodeoxynucleotides designed by Primer3 software (v.0.4.0). The expression of ribosomal protein L13A (RPL13A) was used as a reference gene. The primer sequences and positions into the cDNA are summarized in Table 1. The RNA concentration and its integrity were confirmed by standard procedures. The PCR reaction was performed in duplicate, by adjusting the annealing temperature to 60 °C and in a final volume of 10 μl.
The mitochondrial DNA (mtDNA) quantification was also performed by RT-qPCR with the ThermOne™ RT-PCR Premix (RBC, Taipei, Taiwan). The total cellular DNA extracted using standard procedures was used as template and was amplified with specific oligodeoxynucleotides for MTCO2 and succinate dehydrogenase subunit A (SDHA), as previously reported [17] (link). MtDNA copy number per cell was calculated using SDHA amplification as a reference for nuclear DNA content.
The mitochondrial DNA (mtDNA) quantification was also performed by RT-qPCR with the ThermOne™ RT-PCR Premix (RBC, Taipei, Taiwan). The total cellular DNA extracted using standard procedures was used as template and was amplified with specific oligodeoxynucleotides for MTCO2 and succinate dehydrogenase subunit A (SDHA), as previously reported [17] (link). MtDNA copy number per cell was calculated using SDHA amplification as a reference for nuclear DNA content.
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