The PCR primer sequences for the iNOS and γ-GCS target genes were designed and synthesised by Jiamay Biotech Co. Ltd. The upstream and downstream primers for iNOS were 5 ‘ -ACACCGATTCCACTCAACTA-3 ‘ and 5’-ACCACCTGTTAGTTCAAGCC-3’, respectively; the upstream and downstream primers for γ-GCS were 5’-GCATTCATTTCACCCTGTTCT-3’ and 5’-ACAAAGAGCCCTGACCTAATG-3’, respectively; the lengths of the PCR products were 159 bp and 132 bp, respectively, with β-actin (ComWin Biotech. Co. Ltd., Beijing, China) used as an internal control. Using an ultrapure RNA Kit (CWbio Co. Ltd, Cat#CW0581), the total RNA was extracted from tissue samples. 5 μΕ RNA were used to perform electrophoresis in 1% agarose gel. For the first strand, a HiFi-MMLVcDNA Synthesis Kit (CWbio Co. Ltd, Cat#CW0744) was used for reverse transcription, with UltraSYBR Mixture (with Rox) (CWbio. Co. Ltd, Cat#CW0956) used for amplification.
The amplification protocol was as follows: 95°C for 10 min (95°C for 15 s + 60°C for 60 s) for 40 cycles. The Light Cycler-480II fluorescence quantitative PCR instrument (Idaho Technology, Inc., Utah, USA) was used for measurements, and the 2AACT method was used for the relative quantitative data analysis.
The amplification protocol was as follows: 95°C for 10 min (95°C for 15 s + 60°C for 60 s) for 40 cycles. The Light Cycler-480II fluorescence quantitative PCR instrument (Idaho Technology, Inc., Utah, USA) was used for measurements, and the 2AACT method was used for the relative quantitative data analysis.