Rt pcr kit

Real-time fluorescence quantitative PCR (RT‒PCR): RT‒PCR was performed according to the RT‒PCR kit of Yeasen Biotechnology Co., Ltd., Shanghai, China (No. 11884ES50). The instrument was a StepOnePlus. The specific steps of the test were as follows: pre-denaturation at 95°C for 5 min followed by 40 cycles of 95°C denaturation for 10 s and 60°C annealing/extension for 30 s. The reaction system is presented in Table 5 below. The primers were synthesized by Suzhou Jinweizhi Bioengineering Company. The gene sequence is presented in Table 3. The relative expression was calculated using the 2 – ΔΔCT method.Table 3

Primer sequences for fluorescence-based quantitative real-time PCR.

Table 3

Genes	GenBank number		Primer sequences (5′→3′)	Amplicon size (bp)
β-actin	NM_205518.2	F	ATCTTTCTTGGGTATGGAGTC	141
		R	TCAGCAATGCCAGGGTA
Nrf2	XM_046921130.1	F	GGTGACACAGGAACAACA	223
		R	AAGTCTTATCTCCACAGGTAG
HO-1	NM_205344.2	F	CTGAAGGAAGCCACCAAG	136
		R	CCAGAGCAGAGTAGATGAAG
SOD	NM_205064.2	F	GCTTGTGGTGTAATTGGAAT	159
		R	AGACAGCAGAGTAGTAATGAG
GCLC	XM_046915268.1	F	AGGCTATGTGTCCGATATTG	100
		R	GTTGTTCTTCAGTGGCTCTA
GCLM	NM_001007953.2	F	GCTGCTAACTCACAATGACC	174
		R	TGCATGATATAGCCTTTGGAC
NQO1	NM_001277620.2	F	CACCATCTCTGACCTCTAC	173
		R	CCGCTTCAATCTTCTTCTG

RNAs were extracted using the TRIzol reagent (Beyotime Company, Beijing, China). cDNA was synthesized from RNA using an RT-PCR kit (Yeasen, China), and qRT-PCR was conducted using the SYBR qPCR Mix (Vazyme, Nanjing). The PCR was performed using an ABI 7900 fluorescence quantitative PCR instrument (ABI, USA). The expression of β-actin was applied to standardize mRNA expression and use the variation = 2^{- ΔΔ CT} method calculation. The primers of NGFR and β-actin were as follows: β-actin: 5′-AGAGCTACGAGCTGCCTGAC-3′(F) and 5′- AGCACTGTGTTGGCGTACAG-3′ (R), NGFR: 5′-CAGGTGTGGATCTTTCGTAATCA-3′ (F) and 5′-GTCCGGGTGTGGTAAAACAGG-3′ (R), HO-1:5′-AAGACTGCGTTCCTGCTCAAC-3′ (F) and 5′-AAAGCCCTACAGCAACTGTCG-3′ (R), and FTH: 5′-CCCCCATTTGTGTGACTTCAT-3′ (F) and 5′-GCCCGAGGCTTAGCTTTCATT-3′ (R). Initiating with an initial denaturation stage lasting 10 min at 95 °C, the process progressed through 40 cycles of denaturation at 95 °C for 10 s, annealing at 52.8 °C or 56.2 °C for 15 s, and extension at 72 °C for 20 s. The temperature gradient ranged from 72 °C to 95 °C, increasing in increments of 1 °C per step; the gain calibration was set before the first run.

Cellular or exosomal total RNA was extracted utilizing ISOLATION TRIzol buffer® (Multi Sciences, Hangzhou), followed by cDNA synthesis from the reverse-transcribed RNA using the RT-PCR Kit (Yeasen, China) following the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed using the PerfectStart® Sybr qPCR Mix (Vazyme, Nanjing). Expression levels were determined using the 2^−ΔΔCt method. The primers for miR-494-3p and U6 were as follows.

Gene	Forward seq	Reverse seq
U6	5′-AGGCTTGCTGCTCGGCAGTTGAT-3′	5′-AAGCTAGCTGATCGATCGCTCT-3′
mi-494-3p	5′-TGAAACATACACGGGAACCA-3′	5′-ATGCTGCTAGCTGATCGCTAGCT-3′