Thunderbird sybr qps 201

RNA was isolated from the kidney cortex using RNAiso Plus (Takara, Shiga, Japan) and reverse transcribed with PrimeScript RT Master Mix (perfect Real Time, Takara, Shiga, Japan). A measure of one‐twentieth cDNA was used as a template for subsequent quantification. Polymerase chain reaction (PCR) was run on real‐time PCR: CFX96 System (Bio‐Rad Laboratories, Inc., Hercules, CA), using THUNDERBIRD SYBR QPS‐201 (Toyobo, Osaka, Japan). Real‐time PCR was used to measure the mRNA levels of AKI biomarkers, such as kidney injury molecule‐1 (KIM‐1), liver‐type fatty acid‐binding protein (L‐FABP), NGAL, MCP‐1, and its receptor CCR2, as well as M1 and M2 macrophage markers, to show the correlation with acute‐phase serum creatinine (48 h after bilateral IRI) and the extent of kidney fibrosis. Relative expression levels were calculated using β‐actin mRNA as reference. Table 1 shows the primers for quantification.
Tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), inducible nitric oxide synthase (iNOS), and CD86 used as M1 macrophage markers, and arginase 1, CD163, and CD206 (mannose receptor) were used as M2 macrophage markers.

RNA was isolated using RNAiso Plus (Takara Bio Inc., Shiga, Japan) based on the manufacturer's instructions. Reverse transcription was performed using the PrimeScript RT Master Mix (Takara Bio Inc.). First‐strand cDNA was used to determine the relative mRNA expression with THUNDERBIRD SYBR QPS‐201 (Toyobo Co., Ltd., Osaka, Japan) on the CFX96 System (Bio‐Rad Laboratories, Inc., Hercules, CA, the USA). We have only done up to 39 cycles in RT‐PCR. The expression level of each gene was normalized by β‐actin. All measurements were performed in triplicate, and three independent experiments (n = 3) were conducted. Table 1 shows the primer sequences used in qPCR.

Total RNA was extracted using RNAiso Plus (Takara Bio Inc., Shiga, Japan) according to the manufacture’s protocol. The cDNA was synthesized using Prime Script RT master mix (TaKaRa Bio Inc.). To confirm expression of AhR and β2-AR, the primers shown in Table 1 were used. Quantitative real-time RT-PCR analysis of myostatin, atrogin-1, MuRF-1, and GAPDH was performed in a CFX96 System (Bio-Rad Laboratories. Inc., USA) with THUNDERBIRD SYBR QPS-201 (Toyobo Co. Ltd., Japan). The primers used are shown in Table 2. The threshold cycle (Ct) values for each gene amplification were normalized by subtracting the Ct value calculated for GAPDH.Table 1

Primers in PCR.

Target gene	Forward	Reverse
Ahr	5′-CACAGAGTTAGACCGCCTGG-3’	5′- TTCAGCGCCTGTAACAAGAAC-3’
β2-AR	5′-CTGGTTGGGCTACGTCAACT-3’	5′- CTTCCTTGGGAGTCAACGCT-3’

Table 2

Primers in real time RT-PCR.

Target gene	Forward	Reverse
MuRF-1	5′-GACTCCTGCAGAGTGACCAAG-3’	5′-CTTCTACAATGCTCTTGATGAGC-3’
Atrogin-1	5′-CAGAGAGGCAGATTCGCAAG-3’	5′-GGTGACCCCATACTGCTCTC-3’
Myostatin	5′-CCAGGCACTGGTATTTGGCA-3’	5′-AAGGGATTCAGCCCATCTTCTC-3’
GAPDH	5′-CATGGCCTTCCGTGTTCCTA-3’	5′-CCTGCTTCACCACCTTCTTGAT-3’