Total RNA was extracted from 1 × 107 S2 cells subjected to RNA interference and paraquat treatment using the RNeasy mini kit (Qiagen). Extracted total RNA was used to generate cDNA with random hexamers using the ImProm-II Reverse Transcription System (Promega). Generated cDNA was used as the template in a real-time quantitative PCR assay carried out in a stratagene MX3005P real-time thermocycler. Analysis was performed using Absolute SYBR green ROX master mix (Fisher Scientific). Relative fold change in gene expression was determined by the comparative quantification (2−ΔΔCT) method of analysis [100 (link)]. Taf1 was used to normalize cDNA amounts in the comparative analysis. The primer sets used in the PCR reactions are listed in Additional file 4 : Table S1.
Absolute sybr green rox master mix
Manufactured by Thermo Fisher Scientific
Absolute SYBR green ROX master mix is a ready-to-use solution for real-time PCR that contains SYBR Green I dye and ROX passive reference dye. It is designed to enable sensitive and reproducible quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Improm 2 reverse transcription system, by Promega (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Universal probe library, by Roche (2 mentions)
Cfx96 touch real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Direct zol miniprep kit, by Zymo Research (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (1 mentions)
4 protocols using absolute sybr green rox master mix
1
Gene Expression Analysis of S2 Cells
2
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from 20 to 30 wing discs isolated from wandering third instar larvae using the RNeasy mini kit (Qiagen). cDNA was generated from total RNA using the ImProm-II Reverse Transcription System (Promega) with random hexamers. The cDNA was used as template in a quantitative real-time PCR (qPCR) assay. The analysis was performed using ABsolute SYBR Green ROX master mix (Fisher Scientific) and carried out in a Stratagene Mx3005P real-time thermocycler. Primers used for analysis are given in Supplemental Table S1 . Taf1 and Pgk were used to normalize cDNA amounts in the 2−ΔΔCt comparative analysis47 (link).
3
RNA Extraction and qPCR Analysis of CYP1A1 Expression
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SI tissue was taken from LRD or LRD + I3C fed mice, tissue lysis and homogenization was performed with the Precellys®24 homogenizer, and RNA was isolated using the Zymo Research Direct-zol MiniPrep kit according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using Revert Aid reverse transcriptase (Thermo Fisher Scientific, Bonn, Germany). Real-time PCR was performed on a BioRad CFX96 Touch™ Real-Time PCR Detection System using absolute SYBR-green ROX master mix (Thermo Fisher Scientific). Primers were designed using the Universal Probe Library (Roche Applied Science, Mannheim, Germany): cyp1a1 fwd: 5′-CCTCATGTACCTGGTAACCA-3′, cyp1a1 rev: 5′-AAGGATGAATGCCGGAAGGT-3′, GAPDH fwd: 5′-GAGCCAAACGGGTCATCA-3′, GAPDH rev: 5′-CATATTTCTCGTGGTTCACACC-3′.
4
Quantification of AhR, CYP1A1, and IL-1β
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BMDC and BMMΦ were stimulated with LPS (1 μg/ml) or 3MC (10 μM) for 3 h. RNA extractions were carried out using the RNeasy Fibrous tissue mini kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 μg of total RNA using Revert Aid reverse transcriptase (Thermo Fisher Scientific, Bonn, Germany). Real time PCR was performed on an SDS7300 cycler (Applied Biosystems, Foster City, CA) using absolute SYBR-green ROX master mix (Thermo Fisher Scientific). Primers were designed using the Universal Probe Library (Roche Applied Science, Mannheim, Germany), primers for cyp1A1 were described in65 (link), primers for AhRR were described in54 (link). Primers for EGFP and IL-1β were: EGFPfwd: 5′-aagggcgaggagctgttcac-3′ and EGFPrev: 5′-ttgtgccccaggatgttgcc-3′; IL-1βfwd: ttgacggaccccaaaagat and IL-1βrev: gaagctggatgctctcatca.
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