Thermoscript rt qpcr kits

Total RNA was collected from kidney tissues, THP-1 and RAW264.7 cells, using TRIzol reagents (Invitrogen). First-strand cDNA was synthesized using the Thermoscript RT-PCR synthesis kit (Fermentas, Pittsburgh, PA, USA) according to the manufacturer's instructions. Real-time quantitative PCR analyses for mRNA were performed by using Thermoscript RT-qPCR kits (Fermentas, Pittsburgh, PA, USA) in an ABI PRISM StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The products were used as templates for amplification using the SYBR Green PCR amplification reagent (Qiagen, Valencia, CA, USA) and gene-specific primers. Relative expression levels were calculated according to the standard 2^−ΔΔCt method (21 (link)). The forward and reverse primers used for PCR were as follows:
IL-9 (human) forward: 5′-ATGGTCCTTACCTCTGCCCT-3′; reverse: 5′-GGCACTTGGAAGCTGGATCT-3′; IL-9 (mouse) forward: 5′-TCTTCAGTTCTGTGCTGGGC-3′; reverse: 5′-CAGCTGCATTTTGACGGTGG-3′;glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 5′-CCCAGCAAGGACACTGAGCAAG-3′; and GAPDH reverse: 5′-GGTCTGGGATGGAAA TTGTGAGGG-3′. The expression of GAPDH was used as an internal control (22 (link)).

Total RNA was extracted from mouse liver tissues, KCs and RAW264.7 cells using TRIzol reagents (Invitrogen, USA), and the first-strand cDNA was synthesized using Thermoscript RT-PCR synthesis kit (Fermentas, USA) according to the manufacturer’s instructions. Real-time quantitative PCR analyses for mRNA of TERT, TNF-α, IL-1β, NOS2, CCL2, Arg-1, IL-10, Mrc2, CD163, and β-actin were performed by using Thermoscript RT-qPCR kits (Fermentas, USA) in an ABI Prizm step-one plus real-time PCR System (Applied Biosystems, USA). The mRNA level of β-actin was used as an internal control. Primer sequences were listed in Supplementary Table S1. Relative expression levels were calculated according to the standard 2^−ΔΔCt method. All experiments were performed in triplicate and repeated at least three times.

Total RNA was extracted from RAW264.7 cell lines using TRIzol reagent (Invitrogen, USA). Using ThermoScript RT-qPCR synthesis kit (Fermentas, USA) to synthesized cDNA following to the manufacturer's protocol. Real-time quantitative PCR analysis for mRNA of δ-catenin and GAPDH were performed with ThermoScript RT-qPCR kits (Fermentas, USA). GAPDH was used to normalize input RNA. Relative RNA expression level was calculated following the standard 2^-ΔΔCt method. each experiment was performed in triplicate and repeated for at least three times.