Sybr green master with rox kit

RNA was extracted using TRIzol^® (Thermo Fisher Scientific, Inc.) from each group of cells in the logarithmic growth phase. The concentration and purity of RNA were determined using a spectrophotometer at a wavelength of 260 and 280 nm. RT was performed using the All-in-One™ First-Strand cDNA Synthesis kit (GeneCopoeia) under the following reaction conditions: 42˚C for 60 min and 70˚C for 5 min. Primers were designed and synthesized by GeneCopoeia. qPCR was performed using the SYBR-Green Master with Rox kit (GeneCopoeia). The reaction conditions for qPCR were 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec and 72˚C for 30 sec. mRNA expression in each group was quantified using the 2^-ΔΔCq method (13 (link)).

Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Inc.) from fresh GCTB tissues. RT-qPCR was performed using the All-in-One™ First-Strand cDNA Synthesis kit (GeneCopoeia, Inc.). The temperature protocol was as follows: 42°C for 60 min, 70°C for 5 min. The quantitative primer sequences for p62 and GAPDH primer sequences were as follows: p62 forward, 5′-TCAGGCTGTGGATGAAGTGGAA-3′ and reverse, 5′-CAGAAGATGTTTGTGGCGAGGA-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. The 7th and 8th exon sequencing were as follows: Exon 7 primer forward, 5′-AGACCCCTGCAGCCTTAACT-3′ and reverse, 5′-CCAACTCCTAACCTCCCACA-3′; and exon 8 primer forward, 5′-AGTTGAGCAGTGTGAAAAAGA-3′ and reverse, 5′-GCAGGGAGGGGTCAGGAGCGC-3′. RT-qPCR sequencing was performed using the SYBR-Green Master with Rox kit (GeneCopoeia, Inc.). The reaction conditions for qPCR were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The mRNA expression levels in each group were quantified using the 2^−ΔΔCq method (11 (link)). Sequencing of the 7 and 8th exon amplification products was performed.