RT-PCR analysis of genes related to Cell cycle, B cell differentiation, and IgE production was performed by QuantStudio 5 real-time PCR system (Thermo Fisher, Waltham, MA). For each PCR reaction, 12.5 μL maxima SYBR Green/ROX qPCR Master Mix 2x (Thermo Fisher, Waltham, MA) was mixed with 1.8 μL of 0.3 μM target primers and 300 ng of template DNA to make the total volume as 25 μL. The PCR procedure was set as 40 cycles at 25°C for 5 min, 42°C for 60 min and 70°C for 15 min. GAPDH was used as the housekeeping gene. The Delta Ct values for each gene were calculated by normalizing the Ct values with the housekeeping gene. The relative fold change in mRNA expression between different groups was calculated and expressed as 2−ΔΔCT. The primer sequence used are shown in Supplementary Table 1
Sybr green rox qpcr master mix 2x
Manufactured by Thermo Fisher Scientific
Sourced in United States
SYBR Green/ROX qPCR Master Mix (2X) is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains SYBR Green I dye and ROX passive reference dye, as well as all the necessary components for qPCR, including DNA polymerase, dNTPs, and reaction buffer.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Tri reagent, by Merck Group (4 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Maxima first strand cdna synthesis kit for rt qpcr, by Thermo Fisher Scientific (2 mentions)
Quantstudio 1 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Gen5 take3 module, by Agilent Technologies (2 mentions)
Quantstudio 5, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Genezol trirna pure kit, by Geneaid (1 mentions)
8 protocols using sybr green rox qpcr master mix 2x
1
RT-PCR Analysis of Cell Cycle, B Cell, and IgE Genes
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted with TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, with minor modifications. Total RNA isolated from the liver was reverse-transcribed with a RevertAid First Strand cDNA Synthesis kit (#K1621, Thermo Fisher Scientific). The resulting complementary (c)DNA was amplified in a 96-well PCR plate with SYBR Green/ROX qPCR Master Mix (2X) (Thermo Fisher Scientific) on an Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific). Genes levels were normalized to that of β-actin, and the ratio to β-actin was calculated by setting the value of group C as 1. Information on primers is given in Table 2 .
3
Quantitative Real-Time PCR Analysis of Gene Expression
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The total RNA was extracted from the liver tissue using the TRI-Reagent (Sigma-Aldrich, Steinheim, Germany), and the iWAT was extracted with a GENEzol TriRNA Pure Kit (Geneaid Biotech, New Taipei City, Taiwan), both according to the manufacturer’s instructions, and 3 µg of total RNA was reverse transcribed with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). A quantitative real-time PCR was performed according to the SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific, Waltham, MA, USA) in a 25 μL total reaction volume using the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used in this study are presented in Table 5 . The mRNA expression was normalized against the GAPDH gene as a control and expressed as a multiple of change relative to the control.
4
Hepatic Gene Expression Analysis
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Total RNA was extracted from liver tissue using the TRI Reagent (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instructions, and 3 µg of total RNA was reverse-transcribed with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Thermo Scientific, Waltham, MA, USA). A real-time quantitative (q)PCR was performed according to SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific) in a 25 μL total reaction volume using the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). To evaluate gene expressions, real-time RT-PCRs were performed for five genes (FAS, ACC, SREBP-1c, PPAR-α, and CPT1α) using complementary (c) DNA from liver tissue. The β-actin gene was used as an internal control.
5
RNA Extraction and qRT-PCR Analysis Protocol
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Total RNA was isolated from cells using RNeasy mini-kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The total RNA concentration and purity were determined using Nano drop spectrophotometer at 260 nm absorbance. Reverse transcription was conducted with Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoFisher Scientific, Houston, TX) on 1 μg of total RNA in a final reaction volume of 20 μL according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted in a final volume of 25 μL using SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific) for absolute gene quantification. All reactions were performed in duplicates. Values were normalized to actin and relative levels of mRNA expression were calculated by the 2−ΔΔCT method. The primer sequences used in this study are listed in Supplemental Table 1 . Data are represented as mean or mean of log10 values ± standard deviation of 3 biological replicates.
6
Ileum RNA Extraction and RT-qPCR Analysis
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Total RNA of the ileum was extracted with TRI Reagent® (Sigma-Aldrich, St. Louis, MO, USA), according to the instructions from the vendor. The concentration of RNA was calculated by the ratio of OD 260/280 and was read on a BioTek epoch reader with the Gen5TM Take3 Module (BioTek Instruments, Winooski, VT, USA). The concentration of total RNA was adjusted to 4000 ng/μL, and total RNA was reverse-transcribed with a RevertAid First Strand cDNA Synthesis kit (#K1621, ThermoFisher Scientific, Waltham, MA, USA). The concentration of complementary (c)DNA was adjusted to 50 ng/μL based on results from the BioTek epoch reader with the Gen5TM Take3 Module system. The resulting cDNA was amplified in a 96-well polymerase chain reaction (PCR) plate with SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, Waltham, MA, USA) on a QuantStudio 1 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). The primer sequences are listed in Table 9 . Gene levels were normalized to β-actin, and all groups were compared to the C group by setting the C group to 1.
7
RNA Extraction and qRT-PCR Analysis Protocol
8
Quantitative Gene Expression Analysis
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Total RNA of the liver was extracted with the TRI Reagent® (Sigma-Aldrich, St. Louis, MO, USA), according to instructions from the manufacturer. The quality and quantity of total RNA were evaluated by measuring the OD 260/280 ratio on a BioTek epoch reader with the Gen5TM Take3 Module (BioTek Instruments, Winooski, VT, USA). The concentration of total RNA was adjusted to 4000 ng/µL and then reverse-transcribed with a RevertAid First Strand cDNA Synthesis kit (#K1621, ThermoFisher Scientific, Waltham, MA, USA). The concentration of complementary (c)DNA was calculated by the BioTek epoch reader with the Gen5TM Take3 Module system and adjusted to 50 ng/µL. The resulting cDNA was amplified in a 96-well polymerase chain reaction (PCR) plate using SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific) on a QuantStudio 1 Real-Time PCR System (ThermoFisher Scientific). Gene levels were normalized to β-actin, and the ratio to β-actin was calculated by setting the value of the NC group to 1. Information on primers is given in Table 10 .
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