RNA was extracted using the RNeasy Micro Plus Kit (QIAGEN) according to the manufacturer’s instructions. Retrotranscribed cDNA was obtained from 0.5 to 1 μg of total RNA using the SuperScript VILO retrotranscription kit (Thermo Fisher Scientific).

Real-time qPCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (Applied Biosystems) as the detecting reagent. A total cDNA amount corresponding to 15 ng of starting RNA was used for each reaction. Each sample was analyzed in triplicate and normalized to GAPDH. Relative mRNA quantity was calculated by the comparative cycle threshold (Ct) method using the formula 2−∆Ct [BAZ1B, CCTCGCAGTAAGAAAGCAAAC (forward) and ACTCATCCAGCTCCTTTTGAC (reverse); GAPDH, GCACCGTCAAGGCTGAGAAC (forward) and AGGGATCTCGCTCCTGGAA (reverse); NR2F1, AGAAGCTCAAGGCGCTACAC (forward) and GGGTACTGGCTCCTCACGTA (reverse); NR2F2, GCAAGTGGAGAAGCTCAAGG (forward) and GCTTTCCACATGGGCTACAT (reverse); TFAP2A, GCCTCTCGCTCCTCAGCTCC (forward) and CGTTGGCAGCTTTACGTCTCCC (reverse); and SOX9, AGTACCCGCACTTGCACAAC (forward) and GTAATCCGGGTGGTCCTTCT (reverse)].

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



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