Total RNA was extracted from the lung and kidney with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA concentration was determined by Nanodrop ND1000 at 260 nm (Thermo Scientific). complementary DNA (cDNA) was synthesized using TaqMan reverse transcription reagents and quantified using PC707 (ASTEC Co., Ltd., Fukuoka, Japan). The primers and TaqMan probes for TNF-α, IL-6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were purchased from Sigma Genosys (Sigma-Aldrich, Hokkaido, Japan). The mRNA expression of TNF-α and IL-6 was determined with TaqMan real-time PCR using Lightcycler Nano (Roche Applied Science, Tokyo, Japan). Rat GAPDH was amplified as an internal control, and relative gene expression values were determined using the 2–ΔΔCT method (31 (link)). The following primer sequences were used: TNF-α: 5′-AGGAGAAGTTCCCAAATG-3′, 5′-GTATGAAGTGGCAAATCG-3′, IL-6: 5′-TTGGGACTGATGTTGTTG-3′, 5′-TGAATGACTCTGGCTTTG-3′, GAPDH: 5′-TGAGGCCGGTGCTGAGTATGT-3′, 5′-CAGTCTTCTGGGTGGCAGTGAT-3′.
Pc707
Manufactured by Astec
Sourced in Japan
The PC707 is a laboratory equipment designed for general purpose use. It features a compact and durable construction with basic functionality for laboratory applications. The core function of the PC707 is to provide a reliable platform for various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Merck Group (1 mentions)
Lightcycler nano, by Roche (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Genequant 100, by GE Healthcare (1 mentions)
Abi 7900ht, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
Red imaging system, by Bio-Techne (1 mentions)
Primescript rt master mix, by Qiagen (1 mentions)
5 protocols using pc707
1
Quantitative Analysis of Inflammatory Cytokines
2
Extraction and Quantification of PPAR-α
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Total RNA was extracted using an RNeasy Plus Mini Kit (Qiagen, Shanghai, China) following the manufacturer's protocol. Reverse transcription polymerase chain reaction (RT-PCR) was performed using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Specific primers for PPAR-α were synthesized as follows: PPAR-α forward, 5′-CTGTCGGGATGTCACACAAC-3′ and reverse, 5′-CCGCAAACACCTACTGGATT-3′; GAPDH forward, 5′-CAACGACCACTTTGTCAAGC-3′ and reverse, 5′-TCTTCAAGGGGTCTACATGG-3′. The samples were initially denatured at 95°C for 10 min prior to the thermal cycle (PC-707, ASTEC, Fukuoka, Japan). The thermal cycler for PCR was as follows: 95°C for the 30 s, 63°C for 30 s, and 72°C for 1 min for 35 cycles. The PCR products were separated on a 2% agarose gel and visualized under LED 100 illumination (AMZ System Science, Osaka, Japan).
3
Quantifying Inflammatory Markers in Tissues
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Total RNA was extracted from the kidney, lung, and heart with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA concentration was determined by the absorbance read at 260 nm (GeneQuant 100, GE Healthcare UK Ltd, Chalfont St Giles, Buckinghamshire, UK). The primers and TaqMan probes for tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were purchased from a commercial laboratory (Applied Biosystems, Foster City, CA, USA). The mRNA expressions of TNF-α, IL-1β, and IL-6 were determined by real-time polymerase chain reaction (PCR). cDNA was synthesized using TaqMan reverse transcription reagents (Applied Biosystems, Roche Molecular Systems, Inc., Branchburg, NJ, USA) and quantified using a thermal cycler (PC707, ASTEC Co., Ltd., Minato-ku, Japan). TaqMan real-time PCR was performed using an ABI 7900HT (Applied Biosystems, Foster City, CA, USA). TaqMan rat GAPDH was used as an internal control and relative gene expression values were determined using the 2−ΔΔCT method [21 ].
4
Quantitative RT-PCR Analysis in Zebrafish
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Total RNA was isolated from whole zebrafish embryos using QIAzol Lysis Reagent (Cat. No. 79306; Qiagen, Venlo, Netherlands). The RT reaction was performed with 300 ng of total RNA prepared from each sample using an Omniscript RT Kit (Ca. No. 205111; Qiagen) with a mixture of oligo(dT) and random primers. The RT product (1 µL) was subjected to PCR amplification (15 µL) using a Taq PCR Core Kit (Ca. No. 201223; Qiagen) in a standard thermal cycler PC707 (ASTEC, Fukuoka, Japan). Thermal cycling conditions were as follows: 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 60 s.
The following primers were used: vegfaa, fwd 5'-CTCCTCCATCTGTCTGCTGTAAAG-3' and rev 5'-CTCTCTGAGCAAGGCTCACAG-3' (29 cycles, product size: 490 bp); nos2a, fwd 5'-GTGTTCCCTC AGAGAACAGAT-3' and rev 5'-GATCAGTCCTTTGAAGCTGAC-3' (35 cycles, product size: 822 bp); and elfa, fwd 5'-CTTCTCAGGCTGACTGTGC-3' and rev 5'-CCGCTAGCATTACCCTCC-3' (29 cycles, product size: 358 bp). PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized with SYBR Gold Nucleic Acid Gel Stain (Cat. No. S11494; Molecular Probes, Eugene, OR, USA), followed by detection under a UV transilluminator (Red Imaging System; Alpha Innotech, San Diego, CA, USA). PCR bands were quantitated by densitometry using the ImageJ software in a blinded manner and normalized to the levels of elfa.
The following primers were used: vegfaa, fwd 5'-CTCCTCCATCTGTCTGCTGTAAAG-3' and rev 5'-CTCTCTGAGCAAGGCTCACAG-3' (29 cycles, product size: 490 bp); nos2a, fwd 5'-GTGTTCCCTC AGAGAACAGAT-3' and rev 5'-GATCAGTCCTTTGAAGCTGAC-3' (35 cycles, product size: 822 bp); and elfa, fwd 5'-CTTCTCAGGCTGACTGTGC-3' and rev 5'-CCGCTAGCATTACCCTCC-3' (29 cycles, product size: 358 bp). PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized with SYBR Gold Nucleic Acid Gel Stain (Cat. No. S11494; Molecular Probes, Eugene, OR, USA), followed by detection under a UV transilluminator (Red Imaging System; Alpha Innotech, San Diego, CA, USA). PCR bands were quantitated by densitometry using the ImageJ software in a blinded manner and normalized to the levels of elfa.
5
Extracting and Analyzing Zebrafish Embryo RNA
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Total RNA of whole zebrafish embryos was extracted with QIAzol Lysis Reagent (Cat. No. 79306; Qiagen, Venlo, Netherlands), followed by the RT reaction using an PrimeScript RT Master Mix (Cat.
No. RR036A, Takara Bio Inc., Shiga, Japan). PCR was performed using a Taq PCR Core Kit (Ca. No. 201223; Qiagen) in a standard thermal cycler PC707 (ASTEC, Fukuoka, Japan) under the thermal cycling conditions of 94 °C for 30 s, 53 °C for 30 was blindly performed by densitometry using the ImageJ software and normalized to the levels of elfa.
No. RR036A, Takara Bio Inc., Shiga, Japan). PCR was performed using a Taq PCR Core Kit (Ca. No. 201223; Qiagen) in a standard thermal cycler PC707 (ASTEC, Fukuoka, Japan) under the thermal cycling conditions of 94 °C for 30 s, 53 °C for 30 was blindly performed by densitometry using the ImageJ software and normalized to the levels of elfa.
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