Abi q6 fast real time pcr system

Total RNA was extracted from colon cancer lines using Trizol reagent (Magen, R4801-02, Shanghai, China) and 1 µg of total RNA was reverse transcribed using the PrimeScript RT reagent kit (Vazyme, Nanjing, China) to detect relevant mRNAs. Real-time fluorescence quantitative PCR was performed using SYBR Premix Ex Taq II (Vazyme, Nanjing, China) at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The primer sequences used for PCR are shown in Table 1. Changes in relative expression of different samples were calculated by the ABI Q6 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the 2^−∆∆CT method. GAPDH was used as an internal reference gene.Table 1

Primer sequences used for qRT-PCR analyses

Gene	Primer	Sequence
Mouse GAPDH	m-GAPDH-F	CCAGAGCTGAACGGGAAGCTCAC
	m-GAPDH-R	CCATGTAGGCCATGAGGTCCACC
Mouse CXCL1	m-CXCL1-F	AGCTGCGCTGTCAGTGCC
	mCXCL1-R	CAAGCCTCGCGACCATTC
Mouse IL-6	m-IL6-F	CTCCCAACAGACCTGTCTATAC
	m-IL6-R	CCATTGCACAACTCTTTTCTCA
Mouse IL-1β	m-IL1β-F	AAATGCCACCTTTTGACAGTGA
	m-IL1β-R	GGTTTGGAAGCAGCCCTTCA
Mouse TNF-α	m-TNFα-F	GATCGGTCCCCAAAGGGATG
	m-TNFα-R	CCACTTGGTGGTTTGTGAGTG
Mouse P300	m-P300-F	GCCAACATTGGAGGCACTTT
	m-P300-R	GCCAACATTGGAGGCACTTT

Total RNA was extracted from samples by RNAiso Plus (TaKaRa, Cat. No. 9108) according to the manufacturer’s instructions. mRNA expression levels were quantified by quantitative real-time RT-PCR. The reverse transcription reaction was performed using 1 μg of total RNA with a HiScript II 1st Strand cDNA Synthesis Kit ( + gDNA wiper; Vazyme, Cat. No. R212). Quantitative real-time PCR was performed using ChamQ SYBR Color qPCR Master Mix (High ROX Premixed; Vazyme, Cat. No. Q441) at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. PCR was performed on an ABI Q6 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA), and changes in expression were calculated using the 2^-∆∆Ct method. Primers are listed in Table S2.