Total RNA was extracted from the EPCs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed into cDNA according to the instructions of the Easy Script First Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Beijing, China). Specific primers designed for the amplification of caspase-3, Bax, Bcl-2, NF-κB and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were verified by NCBI Blast; primer sequences are listed in Table I . Reactions were carried out on an ABI PRISM 7300 PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR-Green I GoTaq® qPCR Master Mix (Promega, Madison, WI, USA). PCR reactions were performed in a total of 25 µl as follows: an initial cycle at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec, 58°C for 20 sec and 72°C for 30 sec. The gene expression was analyzed in duplicate and normalized against GAPDH. The results of each gene are expressed as relative expression using the ΔCt method.
Abi prism 7300 pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 7300 PCR system is a real-time polymerase chain reaction (PCR) instrument designed for DNA amplification and detection. It is capable of performing quantitative and qualitative PCR analyses.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Easyscript first strand cdna synthesis supermix kit, by Transgene (3 mentions)
Sybr green 1 gotaq qpcr master mix, by Promega (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Thunderbird sybr qpcr mix, by Toyobo (2 mentions)
Nucleospin rna xs kit, by Takara Bio (2 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Fast sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using abi prism 7300 pcr system
1
Quantifying Gene Expression in EPCs
2
Cytokine and Chemokine Gene Expression in CRC
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Total cellular RNA was extracted from surgical specimens of CRC and autologous healthy mucosa (HM) sampled at distance from the tumor and reverse transcribed [26 (link)]. Pre-developed Taqman® assays (Applied Biosystems) were used to quantitatively evaluate the expression of a panel of cytokine and chemokine genes by using ABI Prism 7300 PCR system (Applied Biosystems). Data are reported as relative expression normalized to GAPDH house-keeping gene amplification. Expression of individual genes was analyzed by using the 2−ΔΔcT method [27 (link)].
3
Analysis of visfatin and PPAR-γ expression
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Total RNA was extracted from BeWo cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using the Easy Script First Strand cDNA Synthesis Super Mix kit (Beijing Transgen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol. RT was performed with a total of 20 µl reaction mixture at 42°C for 15 min and 95°C for 3 min. Specific primers designed for the amplification of visfatin, PPAR-γ, and GAPDH were verified by NCBI BLAST (http://blast.ncbi.nlm.gov/Blast.cgi ). The sequences were as follows: Human visfatin, forward, 5′-GCCAGCAGGGAATTTTGTTA-3′ and reverse, 5′-TGATGTGCTGCTTCCAGTTC-3′; human PPAR-γ, forward, 5′-GCCCTTCACTACTGTTGACTTCT-3′ and reverse, 5′-CAGGCTCCACTTTGATTGC-3′; and human GAPDH, forward, 5′-TGAACGGGAAGCTCACTG-3′ and reverse, 5′-GCTTCACCACCTTCTTGATG-3′. Reactions were performed on an ABI PRISM 7300 PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using PowerUp™ SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed with a total of 20 µl reaction mixture at 50°C for 2 min and 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Gene expression levels were analyzed in triplicate and normalized to GAPDH using the 2−∆∆Cq method (29 (link)).
4
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted from the cells using Trizol reagent (15596026, Invitrogen, USA) and then reverse transcribed into cDNA using the PrimeScript RT reagent Kit (RR047A, Takara, Japan) according to the manufacturer's instructions. The RNA was quantitatively analyzed using the Fast SYBR Green PCR kit (Applied Biosystems) and ABI PRISM 7300 PCR system (Applied Biosystems). Each sample was repeated in triplicate, and GAPDH was used as an internal reference gene. The relative expression level of the gene was analyzed using the 2-ΔΔCt method, where ΔCt = CT (target) - CT (interference), ΔΔCt = ΔCt (test) - ΔCt (control), and the average of three repeated experiments was taken. All primers were purchased from Shanghai Sangon Biotech, and their sequences are shown in Supplementary Table 1 .
5
qPCR Analysis of Lipogenic Gene Expression
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Total RNA was extracted from HepG2 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RNA purity was confirmed by measuring the ratio of absorbance at 260 and 280 nm on a spectrophotometer. Reverse transcription of RNA (8 µl) was conducted according to the instructions of the Easy Script First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech, Co., Ltd., Beijing, China). Specific primers (Table I) for the amplification of regulator sterol regulatory element-binding proteins (SREBP-1c), carbohydrate response element-binding protein (ChREBP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were verified by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). qPCR was performed on an ABI PRISM 7300 PCR system (Applied Biosystems, Thermo Fisher Scientific Inc.) using SYBR Green I GoTaq qPCR Master mix (Promega Corporation, Madison, WI, USA). PCR was conducted in a total volume of 25 µl with the following reaction conditions: 1 cycle at 95˚C for 5 min, followed by 40 cycles of 95˚C for 15 sec, 58˚C for 20 sec and 72˚C for 30 sec. The gene expression from each sample was analyzed in duplicate and normalized against GAPDH. The results are expressed as relative gene expression using the 2 -ΔΔCq method (20) .
6
Quantifying Zebrafish Gene Expression
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Total RNA was extracted from zebrafish at 5 dpf using a Nucleospin RNA XS kit (Takara, Kyoto, Japan) according to the manufacturer’s protocol. cDNAs were generated using a ReverTra Ace qPCR RT Kit (Toyobo). qPCR was performed using an ABI Prism 7300 PCR system (Life Technologies, Carlsbad, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo). The thermal cycling conditions were: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. We measured the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (hmgcr), 7-dehydrocholesterol reductase (dhcr7), mbp, and eukaryotic translation elongation factor 1 alpha 1 (ef1a). hmgcr, dhcr7, and mbp mRNA levels were normalized to ef1a mRNA levels to correct for variability in the initial template concentration and the conversion efficiency of the reverse transcription reaction. The primer sequences are shown in Supplementary Table S6 .
7
Quantitative PCR Analysis of Zebrafish Genes
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qPCR analysis was performed as described previously [42 (link),60 (link)] with some modifications. Briefly, total RNA was extracted from Tg (eno2: cerulean) zebrafish using a Nucleospin RNA XS kit (Takara, Kyoto, Japan) according to the manufacturer’s protocol. cDNA was generated using a ReverTra Ace qPCR RT Kit (Toyobo). qPCR was performed using an ABI Prism 7300 PCR system (Life Technologies, Carlsbad, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo). The thermal cycling conditions were: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s. mRNA expression of neurod1, elavl3, eno2 and irx3b was normalized to that of β-actin (actb) to correct for variability in the initial template concentration and reverse transcription efficiency. The primer sequences are shown in Supplemental Table S3 .
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