All in one rt supermix perfect for qpcr

The RNA isolator total RNA Extraction Reagent (Vazyme, R401-01) was utilized to extract total RNA. After the determination of quality, concentration and integrity of the total RNA, the reagent of All-in-one RT SuperMix Perfect for qPCR (Vazyme, R33) was utilized to reversely transcribe total RNA into cDNA. Real-time quantitative PCR analysis was implemented with the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-03). Primer Premier 5 was used for primers design, and the specific primer sequences are exhibited in Table 5. The total volume of the reaction was 20 µL and the amplification conditions were as follows: 40 cycles of 95 °C for 15 s and 60 °C for 1 min. At the end of the PCR reaction, dissolution curve analysis was performed, and the data were converted into Ct values. The expression of the target gene relative to β-actin was calculated according to the equation of R = 2^−ΔΔCt [23 (link)].

Total RNA was isolated from frozen adjacent normal tissues and renal cell carcinoma samples using the TRIzol protocol (Invitrogen; Thermo Fisher Scientific). Total RNA was isolated from ccRCC cells using the quick RNA extraction kit (Yishan biotechnology). cDNA was synthesized using the All‐in‐one RT SuperMix Perfect for qPCR (Vazyme, Shanghai, Chna). Finally, qRT‐PCR was performed using the ChamQ Universal SYBR qPCR master Mix (Vazyme), primers and diluted cDNA in a Bio‐Rad CFX96 real‐time system according to the manufacturer's protocols. The mRNA expression levels of the genes of interest were first normalized to GAPDH expression, and then the ΔΔCq method was applied to calculate the relative mRNA expression levels of each gene. The primers used in this study were as follows:
RCC1 forward: GGCTTGGTGCTGACACTAGGC.
RCC1 reverse: CCTCCACTGATGTGTCCCTTC.
GAPDH forward: GCACCGTCAAGGCTGAGAAC.
GAPDH reverse: TGGTGAAGACGCCAGTGGA.
EZH2 forward: AGGACGGCTCCTCTAACCAT.
EZH2 reverse: CTTGGTGTTGCACTGTGCTT.

RNA was extracted from G. sessile mycelium of experimental (E) and control (C) groups and used for RT-qPCR. Fungal 18S rRNA was used as an internal reference gene [45 (link), 46 (link)], and Gene-specific primers for RT-qPCR are shown in Additional file 1: Table S4. Three technical repeats were performed. The reaction consisted of the following two steps: RT-PCR (reverse transcription PCR) was performed using the All-in-one RT SuperMix Perfect for qPCR (Vazyme, Jiangsu, China), and the qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme), RT-PCR and qPCR procedures followed the kit steps respectively. The RT-qPCR data were analyzed by the comparative delta-delta Ct method (2^−ΔΔCt) for a relative quantification of the amplicons [47 (link)].