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5 protocols using gene express 2.0 program

1

Quantitative Analysis of Adipogenic Transcription Factors

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The cells were homogenized, and the total RNA was isolated using a Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was obtained using the isolated total RNA (1 μg), a d(T)16 primer, and avian myeloblastosis virus reverse transcriptase (AMV-RT). The relative gene expression was quantified using real-time PCR (Real-Time PCR System 7500, Applied Biosystems, Foster city, CA, USA) with a SYBR green PCR master mix (Applied Biosystems, Foster city, CA, USA). The forward and reverse primers were as follows: PPARγ, 5′-ATCGAGTGCCGAGTCTGTGG-3′ and 5′-GCAAGGCACTTCTGAAACCG-3′; C/EBPα, 5′-GGAACTTGAAGCACAATCGATC-3′ and 5′-TGGTTTAGCATAGACGTGCACA-3′; C/EBPβ, 5′-GGGGTTGTTGATGTTTTTGG-3′ and 5′-CGAAACGGAAAAGGTTCTCA-3′; C/EBPδ, 5′-GATCTGCACGGCCTGTTGTA-3′ and 5′-CTCCACTGCCCACCTGTCA-3′; GAPDH, 5′-GACGGCCGCATCTTCTTGT-3′ and 5′-CACACCGACCTTCACCATTTT-3′.
The gene Ct values of PPARγ, C/EBPα, C/EBPβ, and C/EBPδ were normalized using the Gene Express 2.0 program (Applied Biosystems, Foster city, CA, USA) to the Ct value of GAPDH.
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2

Quantifying Cytokine Expression in RAW 264.7 Macrophages

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RAW 264.7 macrophages (1 × 105 cells/mL) were homogenized, and total RNA was isolated using an easy-BLUE™ total RNA extraction kit (iNtRON Biotechnology Inc., Gyeonggi-do, South Korea). cDNA was obtained using isolated total RNA (1 μg), d(T)16 primer, and avian myeloblastosis virus reverse transcriptase (AMV-RT). Relative gene expression was quantified with real-time PCR (Real Time PCR System 7500, Applied Biosystems, CA, USA) with SYBR green PCR mast mix (Applied Biosystems, CA, USA). The gene Ct values of IL-6 and TNF-α were normalized with the gene express 2.0 program (Applied Biosystems, CA, USA) to the Ct values of GAPDH. Values are presented as mean ± S.D. of three independent experiments.
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3

Quantifying Gene Expression in Cells

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After homogenization of the cells and tissues, total RNA was isolated with Easy-Blue Reagent (Intron Biotechnology Inc, Seongnam, Republic of Korea) according to the manufacturer’s instructions. Quantification of total RNA was conducted with an Epoch microvolume spectrophotometer system (BioTek Instruments Inc., Winooski, VT, USA). cDNA was obtained using isolated total RNA (1 μg), d(T)16 primer, and AMV reverse transcriptase. Relative gene expression was quantified using real-time PCR technique (Real Time PCR System 7500, Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems) and oligonucleotide primers (Table 1) purchased from Bioneer. The threshold cycle (Ct) values of PPARγ, C/EBPα, and SREBP1 genes were normalized to the Ct values of GAPDH using gene express 2.0 program (Applied Biosystems). We used the comparative Ct method to calculate the fold changes in gene expression.
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4

RNA Extraction and Real-Time PCR for Cytokine Analysis

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RAW 264.7 macrophages (1 × 105 cells/mL) were homogenized, and total RNA was isolated using an easy-BLUE™ total RNA extraction kit (iNtRON Biotechnology Inc., Gyeonggi-do, South Korea). cDNA was obtained using isolated total RNA (1 μg), d(T)16 primer, and avian myeloblastosis virus reverse transcriptase (AMV-RT). Relative gene expression was quantified with real-time PCR (Real-Time PCR System 7500, Applied Biosystems, CA, USA) with SYBR green PCR mast mix (Applied Biosystems, CA, USA). The gene Ct values of IL-6 and TNF-α were normalized with the gene express 2.0 program (Applied Biosystems, CA, USA) to the Ct values of GAPDH.Values are presented as mean ± SD of three independent experiments. Oligonucleotide primers were got from Bioneer (Daejeon, Republic of Korea) (Table 2).
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5

RNA Extraction and Quantitative Gene Expression

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After homogenization of the cells and tissues, total RNA was isolated with Easy‐Blue Reagent (Intron Biotechnology Inc.) according to the manufacturer's instructions. Quantification of total RNA was performed with an Epoch microvolume spectrophotometer system (BioTek Instruments Inc.). cDNA was obtained using isolated total RNA (1 µg), d(T)16 primers and AMV reverse transcriptase. Relative gene expression was quantified using real‐time PCR technique (Real‐Time PCR System 7500, Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems) and oligonucleotide primers (Table 1) purchased from Bioneer. The threshold cycle (Ct) values of genes were normalized to the Ct values of GAPDH using the gene express 2.0 program (Applied Biosystems). We used the comparative Ct method to calculate fold changes in gene expression.
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