Sybr green 1 fluorescence detection kit

ITGA5 mRNA expression in the adjacent tissues, gastric cancer tissues, and gastric cancer cell lines was detected by qRT-PCR. Total RNA was extracted using the Nucleozol Total RNA Extraction Kit (MACHEREY-NAGEL, Germany), and its concentration and purity were detected by Nanodrop ND-2000. The expression levels of different mRNAs were detected by Takara Biotechnology Ltd. RNA was reverse transcribed using a reverse transcription kit. qRT-PCR was performed using SYBR Green I fluorescence detection kit (Takara, China), and the quantitative analysis was performed using 7500 real-time PCR system. The primers were synthesized by Shanghai Bioengineering Technology Co., Ltd., and the sequences were as follows: target gene ITGA5, forward: 5′-GGCTTCAACTTAGACGCGGAG-35′- CACCCTGTTGCTGTAGCCAAA-3′; reverse: 5′-TGGCTGGTATTAGCCTTGGGT-3′; internal reference GAPDH, forward: 5′- TGACTTCAACAGCGACACCCA-3^″; reverse: 5′- CACCCTGTTGCTGTAGCCAAA-3′; The reaction conditions were as follows: 95°C pre-denaturation for 30 s, 95°C for 10 s, 60°C for 30 s, 35 cycles. The experiment was performed using 3 replicates.

TRIzol reagent was used to extract total RNA from tissues, and cDNA was obtained using a reverse transcription kit from Takara Biotechnology Co., Ltd. SYBR-Green I fluorescence detection kit and a 7500 Real-Time PCR System were used for qRT-PCR to detect ITGA5 mRNA expression. The reaction conditions were as follows: 95°C, 30 s; 95°C, 5 s; 63°C, 30 s; 40 cycles. The primers were synthesized by Sangon. The GAPDH primer sequence was as follows: F 5′-TGACTTCAACAGCGACACCCA-3′, R 5′-CACCCTGTTGCTGTAGCCAAA-3′, while the ITGA5 primer sequence was as follows: F 5′-GGCTTCAACTTAGACGCGGAG-3′, R 5′-TGGCTGGTATTAGCCTTGGGT-3′.