Iq5 real time pcr

RNA was extracted from these four cells using the Redzol reagent (Sebasun, Beijing, China). Synthesis of cDNA using a reverse transcription kit (Toyobo Biotech Co., Ltd., Shanghai, China), and then the mRNA expression level was detected by iQ5 Real-Time PCR (Applied Biosystems, USA). The PCR reaction program was (95°C for 60 s, then 95°C for 15 s, 60°C for 60 s, 40 cycles). All PCR experiments were repeated three times. Finally, all results were normalized by GAPDH. Relative expression levels for these genes were calculated using 2 -ΔΔCt. The primer sequences were: GAPDH, F-5′-TCAGCAATGCCTCCTGCAC-3′, R-5′-TCTGGGTGGCAGTGATGGC-3′. NCKAP1, F-5′-TGCTGTAGAAACCCGCAACA-3′, R-5′-TCTGGGTGGCAGTGATGGC-3′.

We extracted total RNA from intestinal tissues using TriQuick Reagent according to the manufacturer’s instructions (Solarbio, Beijing, China). Next, we synthesized cDNA using a reverse transcription kit (GeneCopoeia, Guangzhou, China). We then conducted qRT‒PCR amplification using iQ5 Real-Time PCR (Applied Biosystems, USA) with 2 × SYBR Green qPCR Master Mix (No ROX) (Servicebio, Wuhan, China). Finally, we calculated the relative mRNA expression of the genes using the 2-ΔΔCT method.