Cdna synthesis supermix kit

Total RNA was extracted from GBM cells by using TRIzol™ reagent, according to the manufacturer’s instructions. The RNA concentrations were examined by a spectrophotometer (NanoDrop 1000, Thermo). The first-strand cDNAs were synthesized using a cDNA Synthesis SuperMix kit (YEASEN, China, #11123ES10). Each cDNA (2 μL) was amplified using the Hieff qPCR SYBR Green Master Mix (final volume, 20 ml, YEASEN, China, #11203ES03) and then analysed on the Applied Biosystems 7900 Real-time PCR Detection System. Thermal cycling conditions were performed as follows: melting step (95°C for 30 s), annealing step (40 cycles of 95°C for 10 s), and elongation (60°C for 30 s). Actin was used as internal control. Melting curves were used for verifying the specificity of each PCR reaction. The cycle threshold (Ct) was the mean value of three Ct values. The relative expression level of RNA was analyzed by the 2^−ΔΔCt method.
RNA primers are listed as follows:

ECM1 (human-forward): 5′-TGA​ACC​AAA​TCT​GCC​TTC​CTA​AC-3’

ECM1 (human-reverse): 5′-GCT​GGA​CTG​TGG​TAG​GTT​CCA-3’

GAPDH (human-forward): 5′-CGA​AAT​CCC​ATC​ACC​ATC​TTC​CAG​G-3’

GAPDH (human-reverse): 5′-GAG​CCC​CAG​CCT​TCT​CCA​TG-3’.

Total RNA was extracted according to TRIzol reagent (Servicebio, Wuhan, China). cDNA was obtained by reverse transcription using the cDNA synthesis supermix kit (Yeasen, Shanghai, China). The 20 μL qRT-PCR system was established according to the qPCR SYBR Green master mix kit (Yeasen, Shanghai, China). β-actin was served as internal reference. The relative expression levels were calculated according to the 2^−△△Ct method. qPCR was performed with three independent experiments. p-values< 0.05 were regarded as statistically significant. The sh-RNA sequences and primers sequences (5’-3’) were showed in Supplementary Table S1.

RNA was reverse transcribed using the cDNA synthesis SuperMix kit (Yeasen Biotech Co., Ltd., Shanghai, China). RT-qPCR was performed using our previous method (Pan et al., 2022 (link)). Gene-specific primers were listed in Table S9. All reactions included three biological replicates. The relative gene expression level was calculated according to the 2^-△△CT method (Livak and Schmittgen, 2002 (link)), with P. avium actin gene as an internal standard.