Gi 1 evagreen qpcr master mix

Complementary
DNA was synthesized using the reverse transcription method with the
WizScript cDNA Synthesis Kit (Wizbiosolutions). Briefly, 10 μL
of 2× RT master mix was prepared in microtubes by adding a random
hexamer (2 μL), 10× reaction buffer (2 μL), WizScript
RTase (1 μL), 20× dNTP mix (1 μL), RNase inhibitor
(0.5 μL), and RNase-free water (3.5 μL) on ice. After
that, 500 ng of total RNA was incorporated with the 2× RT master
mix. A final volume of 20 μL was made up with RNase-free water,
and the microtubes were centrifuged to remove any air bubbles. The
microtubes were then put in a thermal cycler set at the following
settings: the first step of 10 min at 25 °C, the second step
of 120 min at 37 °C, the third step of 5 min at 85 °C, and
the fourth step of infinite time at 4 °C. The prepared cDNA was
preserved at −20 °C, and qPCR was performed to study the
gene expression using GI I EvaGreen qPCR Master Mix (GeneDirex). Briefly,
10 μL of the master mix was mixed with 0.5 μL each of
the forward and reverse primers, 1 μL of cDNA, and 8 μL
of DNase-free water and put into an iQ5 real-time PCR detection system
(Bio-Rad) at an annealing temperature of 56 °C. The primer sequence
used in this study was reported in an earlier study,^{17 (link)} and HPRT1 was used as an internal control gene. The relative
fold change expression was estimated by the 2^–ΔΔCt method.^{18 (link)}

Trans-Chalcone (Sigma-Aldrich), diclofenac sodium, complete
Freund’s adjuvant, formalin, DMSO, chloroform, isopropanol,
and ethanol were from Sigma-Aldrich, besides TRI-Reagent RT (BioShop
Canada), WizScript cDNA Synthesis Kit (High Capacity) (Wizbiosolutions),
GI I EvaGreen qPCR Master Mix (GeneDirex), Rat PGE2 ELISA Kit (Wuhan
Zokeyo Biotechnology Co., Ltd.), and nitric oxide (NO) assay kit (Solarbio
Life Sciences).