Total RNA was extracted by using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The purity and quality of RNA were confirmed by defining the ratio of absorbance at 260 and 280 nm wavelengths (NanoDrop® ND-1000, Thermo Scientific, Waltham, MA, USA). A sample of 3 µg total RNA was reverse transcribed into cDNA. For mRNA measurement, diluted cDNA was amplified using the Rotor-Gene SYBR Green PCR Kit (Qiagen, Hilden, Germany) in a Rotor-Gene Q 2plex HRM Platform (Qiagen). Reaction conditions were carried out for 35–40 cycles (5 min at 95 °C, 5 s at 95 °C and 10 s at 60 °C). All procedures for RNA extraction and qPCR have been described previously [57 (link),58 (link),59 (link)].
The primers were designed using reported cDNA sequences:1. TNF-α, Forward 5′-CTC TTC TCA TTC CCG CTC GTG-3′ and Reverse 5′-GGA ACT TCT CCT CCT TGT TGG G-3′; 2. IL-1β, Forward 5′-GTT TGA GTC TGC ACA GTT CCC-3′ and Reverse 5′-CAA CTA TGT CCC GAC CAT TGC-3′; 3. IL-6, Forward 5′-TTC TTG GGA CTG ATG TTG TTG AC-3′ and Reverse 5′-AAT TAA GCC TCC GAC TTG TGA AG-3′; 4. β-actin, Forward 5′-GAC CCA GAT CAT GTT TGA GAC CTT C-3′and Reverse 5′-GAG TCC ATC ACA ATG CCW GTG G-3′.
The primers were designed using reported cDNA sequences:
Free full text: Click here
