Complementary DNA (cDNA) was prepared from 1 µg of total RNA from each sample using the High-Capacity cDNA Archive Kit (Life Technologies, Grand Island, NY, USA). Real-time polymerase chain reaction (RT-PCR) was performed on a Roche LightCycler 480 thermal cycler (Roche Diagnostics, Indianapolis, IN, USA) with Roche LightCycler TaqMan Master Kit (Roche Diagnostics) for confirmation of microarray results. The 18S primer/probe was used as an endogenous control for each sample and measured simultaneously with each labeled sample for the purpose of normalization; relative quantification was determined by the comparative CT method.21 (link) Primer/probes of interest (JUNB, FOS, DUSP1, and EGR3) and 18S primer sets were purchased from TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Dimethyl sulfoxide–treated (vehicle) HUVEC at time 0 were used as the comparator for all CDDO-Im–treated samples.
Lightcycler taqman master kit
Manufactured by Roche
Sourced in Germany
The LightCycler TaqMan Master Kit is a reagent kit designed for real-time PCR analysis. It contains all the necessary components, including a master mix, primers, and probes, to perform quantitative PCR experiments.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (7 mentions)
Lightcycler 480, by Roche (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Lightcycler 2.0 carousel based system, by Roche (4 mentions)
High pure rna isolation kit, by Roche (3 mentions)
Lightcycler, by Roche (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Lightcycler 1, by Roche (2 mentions)
Lightcycler software 3, by Roche (2 mentions)
Universal probe library probe, by Roche (2 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Universal probe library, by Roche (2 mentions)
Universal probe library system, by Roche (2 mentions)
Isolate 2 rna plant kit, by Meridian Bioscience (2 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Dnase digestion step, by Qiagen (1 mentions)
37 protocols using lightcycler taqman master kit
1
Evaluating Gene Expression Changes in Endothelial Cells
2
Relative Expression of Chicken Lens Crystallins
3
Quantifying Liver Fibrogenesis and Lipogenesis
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Real-time quantitative polymerase chain reaction (RT-qPCR) of liver tissues was performed to determine the mRNA expression of the most important fibrogenic gene transforming growth factor (TGF)-β1 and the lipogenic gene sterol regulatory element-binding factor-1 (SREBF-1). The liver total RNA content was extracted using the Trizol method and RNeasy mini kit (Qiagen NV, Venlo, the Netherlands). The cDNA was collected after reaction in a PCR machine for 5 minutes at 25°C, 60 minutes at 50°C, and 15 minutes at 70°C, and it was subsequently stored at −20°C. The primers were TGF-β1, forward: 5′-gAgCAACATgTggAACTCTAC-CAg-3′ and reverse: 5′-AAgCCCTgTATTCCgTCTCCT-3′ and SREBF-1, forward: 5′-TTCCATTgACAAggCCATgC-3′ and reverse: 5′-gCCTCATgTAggAATACCCTCCTC-3′. The sample cDNA was diluted, and RT-qPCR was subsequently performed using a Lightcycler TaqMan master kit (Hoffman-La Roche Ltd., Basel, Switzerland) under the following PCR conditions: preincubation at 95°C for 10 minutes, one cycle; amplification at 95°C for 10 seconds, 60°C for 30 seconds, and 72°C for 5 seconds, 50 cycles; cooling at 40°C for 30 seconds, one cycle.
4
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In experiments investigating SFN gene expression coding for 14-3-3 σ, a DNase digestion step (Qiagen Hilden, Germany) was included during RNA isolation in order to minimize DNA contaminations as no intron-spanning primers could be designed for this gene. One µg RNA was reversely transcribed with Expand Reverse Transcriptase (Roche, Penzberg, Germany) and poly(dT) oligonucleotide primers (Eurofins Genomics, Ebersberg, Germany). Quantitative real-time PCR was performed using the LightCycler TaqMan Master Kit (Roche, Penzberg, Gemany) together with the Universal Probe Library System. RT-PCR primers were designed using the open software “Universal Probe Library Assay Design Center” from Roche [60 ] and intron-spanning primers selected whenever possible (listed in Table 1 ). Gene expression was normalized to hypoxanthine phosphoribosyltransferase (HPRT) expression.
5
Quantitative Real-Time PCR Protocol
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Total RNA was extracted using TRIzol reagents (Invitrogen) for heart tissues or PureLink RNA Mini Kit (Ambion) for cultured cells according to manufacturer's instructions. RNA samples were subjected to DNase treatment to remove genomic DNA using TURBO DNA-free Kit (Ambion) or PureLink DNase Set (Ambion) and were reverse-transcribed using SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative real-time PCR was performed using Universal Probe Library (UPL, Roche) and Light Cycler TaqMan Master kit (Roche). Relative expression levels of the target genes were normalized to the expression of internal control gene using comparative Ct method. Primer sequences and the corresponding UPL numbers were designed with online program provided by Roche.
6
Quantitative Analysis of CYP1A1 Expression
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HepG2, Caco-2 and MCF-7 cells were exposed to AhR agonists or coffee in either PBS or AHS for 4 h, in 12-well plates. Three wells of cells received each treatment. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and was treated with RNase-free DNase (Promega). Reverse transcription was performed using the extracted total RNA, random hexamer primers (Amersham, Uppsala, Sweden), dNTPs, RNAsin (Promega), and Moloney murine leukemia virus reverse transcriptase (Promega). The cDNA thus obtained was subjected to quantitative real-time PCR, which was performed using a LightCycler TaqMan Master kit (Roche Applied Science, Penzberg, Germany) and a LightCycler 480 (Roche Applied Science). The PCR conditions were set according to the manufacturer’s instructions. The primers for human CYP1A1 (5′-TCCAAGAGTCCACCCTTCC-3′ and 5′-AAGCATGATCAGTGTAGGGATCT-3′ ) and the probe used for detection (#83; Roche Applied Science) were determined using the Roche Applied Science Universal ProbeLibrary Assay Design Center (http://www.roche-applied-science.com ). For each sample, real-time PCR amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed simultaneously using the Universal ProbeLibrary Human GAPD Gene Assay (Roche Applied Science), and the CYP1A1/GAPDH ratio was calculated.
7
Quantification of RXRα Expression in Umbilical Cord Samples
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Five micrograms of total RNA from umbilical cord samples were synthesized to cDNA using the Transcriptor First-Strand cDNA synthesis kit (Roche) following the manufacturer’s instructions. Assay efficiency was calculated using a dynamic range of cDNA dilution series (1:1, 1:2, 1:10, 1:100, 1:1,000, and 1:10,000). Quantitative real-time PCR was performed using the LightCycler TaqMan Master Kit (Roche), and TaqMan gene expression assay probes for RXRα and β-actin were purchased from Thermo Fisher Scientific. qPCR conditions for RXRα gene expression were the following: for amplification, 10 min at 95°C followed by 45 cycles of 10 s at 95°C, 30 s at the primer annealing temperature (60°C), and 10 s at 72°C. qPCR assays were performed three times in duplicate using a LightCycler Nano Instrument (Roche). Gene expression data were normalized using β-actin expression. Fold change in expression was calculated using the 2−ΔΔCt method.
8
Gene Expression Analysis in Lilium
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The expression of LlACS (GenBank acc. no KF573522), LlACO (GenBank acc. no KF573523), LlZEP, LlIDL (GenBank acc. no KT716180), LlHSL, and LlMPK6 genes was analyzed in relation to LlACT (GenBank acc. no KP257588), which was used as a reference endogenous control for normalization purposes. RT-qPCR was performed with a LightCycler 2.0 Carousel-Based System (ROCHE Diagnostics GmbH, Germany) and a LightCyclerTaqMan Master Kit (ROCHE Diagnostics GmbH, Germany), using primers and UPL probes designed previously [5 (link),7 (link)]. The list of gene-specific and reference primers used is presented in Supplementary Figure S4 . The qPCR mixture preparation and conditions were as described previously [7 (link)]. Reaction efficiencies (>99%) were calculated based on the standard curves from serial dilutions of cDNA templates, and relative expression values were obtained by LightCycler Software 4.1 (Roche Diagnostics GmbH, Mannheim, Germany). The data are presented as means ± standard deviation (SD) of three biological repeats obtained from three independent experiments.
9
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was treated with DNase and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Real-time quantitative PCR was performed using the Universal Probe Library (UPL) (Roche) and Light Cycler TaqMan Master kit (Roche). Relative levels of gene expression were normalized to the Gapdh gene expression using the comparative Ct method. Primer sequences and the corresponding UPL numbers were designed using an online program provided by Roche. Primer sequences are provided in Supplementary Table 1 .
10
Quantitative RT-PCR for Gene Expression
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Total RNA extraction and DNase treatment were performed by using SV total RNA Isolation Kit (Promega). RNA was DNase treated and reverse transcribed by using QuantiTect Reverse Transcription Kit (Qiagen). Real‐time quantitative PCR was performed by using Universal Probe Library (UPL) (Roche Molecular Diagnostics) and Light Cycler TaqMan Master kit (Roche). Relative levels of gene expression were normalized to the mouse 28S rRNA expression by using the delta delta CT method according to the manufacturer's instructions and shown as fold increase to sham‐operated mouse. Primer sequences and UPL number were listed in Table 1 .
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