Sybr green qpcr super mix kit

TRIZOL reagent (Thermo Fisher Scientific, Inc.) was selected to obtain total RNA in tissues and cells, and the purity and concentration of total RNA were determined via a spectrophotometer (Eppendorf) [19 (link)]. According to the requirements of the instructions, the Reverse Transcription Kit (Promega Corporation) was used to reverse transcribe RNA into cDNA. Then the SYBR®GREEN qPCR Super Mix Kit (Vazyme) was used to configure the reaction system, and the RT-qPCR assay was performed through the 7500 sequences detection system (Thermo Fisher Scientific, Inc.). The reaction system was 10 µL, including 2.5 µL cDNA, 5 µL 2× SYBR GREEN qPCR Super Mix, 0.5 µL primers and 2 µL ddH₂O. Subsequently, GAPDH and U6 were used as endogenous controls, and the data was calculated and processed by 2^−ΔΔCt method. The primer sequences are as follows: MELTF-AS1 forward, 5´-GCGTTCACACTCATTACCC-3´ and reverse, 5´-CTATTCAGACCCCTTCACCC-3´ GAPDH forward, 5´-GAGCCACATCGCTCAGACAC-3´ and reverse, 5´-GCCCAATACGACCAAATCC-3´ U6 forward, 5´-GCTTCGGCAGCACATATACTAAAAT-3´ and reverse, 5´-CGCTTCACGAATTTGCGTGTCAT-3´.

Total RNA was extracted from tissues using TRIzol according to the manufacturer’s protocol. Nuclear and cytoplasmic RNA were isolated from cells using RNA Subcellular Isolation Kit (Active Motif, Shanghai, China). cDNA synthesis was performed using a HiScript Q Select RT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech Co., Nanjing, China). qRT-PCR analysis was performed using SYBR Green qPCR SuperMix Kit (Vazyme Biotech Co., Nanjing, China) according to the manufacturer’s protocol. The expression of the target genes was normalized to that of GAPDH. The primers used for amplification are described in Supplemental Materials and Methods.

Total RNA was extracted from cells with TRIzol reagent (Invitrogen, USA). Nuclear and cytoplasmic RNA were isolated from cells using RNA Subcellular Isolation Kit (Active Motif, USA). Reverse transcription to cDNA was performed using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, USA). qRT-PCR analysis was performed using SYBR Green qPCR SuperMix Kit (Vazyme Biotech, China) according to the manufacturer’s protocol. The primer sequences were listed in Supplementary Table 1.