Qpcr sybr green master mix

Total RNA from tissues was extracted with TRIzol reagent. The RNA was reverse transcribed to cDNA following the instructions of reverse transcription kit PrimeScript^® RT Master Mix Perfect Real Time kit (TaKaRa, Dalian, China). Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using SYBR Green qPCR Master Mix (Yeasen) on an ABI PRISM 7500 fast Sequence Detection System (Applied Biosystems, Foster City, CA, USA) following the protocols. Primers were designed to perform the amplification, and the sequences were as follows: GAPDH, Forward: 5′-TATGATGATATCAAGAGGGTAGT-3′ and Reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′; circ_0078607, Forward: 5′-CGGAATTCTGAAATATGCTATCTTACAGTTTGACCTTGTCTGTGTCAATG-3′ and Reverse: 5′-CGGGATCCTCAAGAAAAAATATATTCACCTCTGAGTAATTTGATGAGAGG-3′; GAPDH as an internal control. The comparative expression level was compared using 2^−ΔΔCt method.

Total RNA was extracted from OS tissues (0.5×0.5×0.3 cm), adjacent tissues (0.5×0.5×0.3 cm) and cells lines (MG63 and SaSO-2, 5×10⁵ cells) using TRIzol^® reagent [Yeasen Biotechnology (Shanghai) Co., Ltd.], according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit [Yeasen Biotechnology (Shanghai) Co., Ltd.] according to the manufacturer's protocol. qPCR was subsequently performed using SYBR Green qPCR Master Mix [Yeasen Biotechnology (Shanghai) Co., Ltd.]. The following primer pairs were used for the qPCR: CLEC3A forward, 5′-CGAGGCACTAAAGTTCACAAGA-3′ and reverse, 5′-CGGAGTTCCTGGGGATAACCA-3′; AKT1 forward, 5′-AGCGACGTGGCTATTGTGAAG-3′ and reverse, 5′-GCCATCATTCTTGAGGAGGAAGT-3′; MCL1 forward, 5′-TGCTTCGGAAACTGGACATCA-3′ and reverse, 5′-TAGCCACAAAGGCACCAAAAG-3′; GLUT1 forward, 5′-GGCCAAGAGTGTGCTAAAGAA-3′ and reverse, 5′-ACAGCGTTGATGCCAGACAG-3′; VEGF forward, 5′-AGGGCAGAATCATCACGAAGT-3′ and reverse, 5′-AGGGTCTCGATTGGATGGCA-3′; β-actin forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; and 40 cycles of 95°C for 30 sec and 60°C for 30 sec. Expression levels were quantified using the 2^−ΔΔCq method (11 (link)) and the internal reference gene β-actin acted as the control.

The mycelia of the corresponding strains were cultured in the PS medium at 28°C for 7 days. The process of collecting mycelia involved filtering the shaken liquid culture through gauze, and then using paper towels to dry the remaining moisture. After grinding the collected mycelia into a fine powder with liquid nitrogen, RNA extraction was performed using Trizol (Sango Biotech, B511311) reagent according to the instructions. Reverse transcription of total RNA was conducted with the PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA, RR047A). SYBR Green qPCR Master Mix (Yeason, 11201ES03) was used to conduct the qRT-PCR assay. The relative expression levels of genes were evaluated by the 2^−ΔΔCt method with the tubulin gene (Uv8b_900) as an endogenous reference. In the supplementary Table S1, the required primers are listed. All assays were independently conducted with three technical replicates and two biological replicates.

According to the instructions provided by RNAiso Plus (Takara, Beijing, China), total RNA were extracted and performed reverse transcription using PrimeScript™ RT Master Mix (Takara, Beijing, China). Subsequently, qPCR was performed utilizing SYBR Green qPCR Master Mix (Cat#11202ES08, Yeason, Shanghai, China). GAPDH was employed as the internal reference gene for mRNAs. The primer sequences utilized in this study are detailed in Table 1. The relative expression levels of each gene were determined using the 2^−(ΔΔCt) method.