Total RNA was extracted by using Trizol reagent according to the manufacturer’s instructions and reverse-transcribed into cDNA. PCR experiments were then conducted with ABI7500 system (Life Technologies, United States) by TB GreenTM Premix Ex TaqTM (Tli RNaseH Plus) and ROX plus kit [Takara Biomedical Technology (Beijing) Co., Ltd., China]. GAPDH was used as a housekeeping gene. The mRNA levels were calculated by using the ΔΔCT method. The sense and antisense primers (TNF-α, IL-1β, IL-6, and GAPDH) as follows were purchased from Sangon Biotech (Shanghai) Co., Ltd. (China). TNF-α: 5′-TACTGAACTTCGGGGTGATTGGTCC-3′ and 5′-CAGCCTTGTCCCTTGAAGAGAAC-3′. IL-1β: 5′-GCACTACAGGCTCCGAGATGAAC-3′ and 5′-TTGTCGTTGCTTGGTTCTCCTTGT-3′. IL-6: 5′-CCGGAGAGGAGACTTCACAG-3′ and 5′-GGAAATTGGGGTAGGAAGGA-3′. GAPDH: 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-CACCACCCTGTTGCTGTAGCC-3′.
Rox plus kit
Manufactured by Takara Bio
The ROX plus kit is a laboratory reagent used for real-time PCR experiments. It contains a reference dye that is used to normalize fluorescence signals during the PCR reaction.
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Lab products found in correlation
2 protocols using rox plus kit
1
Quantification of Inflammatory Cytokines
2
Quantitative Detection of SKP2 and miR-186
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For quantitative detection of SKP2, total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, China). Quantitative real-time PCR analysis was performed using the Premix Ex Taq (Probe qPCR, Takara) and ROX Plus Kit (Takara) following the manufacturer's instructions. The ratio of SKP2 to β-actin was used as the normalized relative level of SKP2 expression. The primers and probe used for SKP2 cDNA detection were 5′-CC TTTCTGGGTGTTCTGGAT-3′ (sense), 5′-CAGCCACCTGT ACATGCTTT-3′ (antisense), and 5′Fam-TGCCCTGCAGAC TTTGCTAAGCA-Tamra 3′ (probe). The primers and probe used for β-actin cDNA detection were 5′-CACTCTTCCA GCCTTCCTTC-3′ (sense), 5′-GGATGTCCACGTCACACTT C-3′ (antisense), and 5′Fam-TGCCACAGGACTCCATGCCC -Tamra 3′ (probe).
For quantitative detection of miR-186, qRT-PCR analysis was performed using the Two Step Stemaim-it miR-186 qRT-PCR Quantitation Kit (Novland, China). We quantified U6 small nuclear RNA (U6 snRNA) as an endogenous control to normalize miRNA level. Relative expression level was analyzed using the 2 -ΔΔCt . Each sample was analyzed in triplicate on the ABI 7500 Fast thermocycler.
For quantitative detection of miR-186, qRT-PCR analysis was performed using the Two Step Stemaim-it miR-186 qRT-PCR Quantitation Kit (Novland, China). We quantified U6 small nuclear RNA (U6 snRNA) as an endogenous control to normalize miRNA level. Relative expression level was analyzed using the 2 -ΔΔCt . Each sample was analyzed in triplicate on the ABI 7500 Fast thermocycler.
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