RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR). RNA extraction of brain subregion samples was conducted with RNeasy Mini Kit (Qiagen) with on column DNase digestion (Qiagen), based on the manufacturer’s protocol. The RNA concentration and quality were measured by NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA). Total 0.6 μg RNA from each sample was reverse transcribed in 20 μl total volume using the iScript cDNA synthesis kit (Bio-Rad).
Gene expression of brain subregions was measured using FastStart Universal SYBR Green Master with Rox (Roche) and analyzed using ABI 7500 RT PCR System (Life Technologies, Carlsbad, CA, USA). Gene expression was normalized to gapdh mRNA. Data are presented as fold-change in gene expression in each group relative to control group. The primers of Asic1a, Asic1b, Asic2a, Asic2b, and Asic4 were adapted from previous study (Schuhmacher and Smith, 2016 (link)). Asic3 primers were designed based on Primer-BLAST (NCBI, NIH). The primer sequence for Asic3 were forward, 5′-TATGTGGCTCGGAAGTGCGGAT-3′, and reverse, 5′-CAGACACAAGTGTCCTTTCGCAG-3′.
Gene expression of brain subregions was measured using FastStart Universal SYBR Green Master with Rox (Roche) and analyzed using ABI 7500 RT PCR System (Life Technologies, Carlsbad, CA, USA). Gene expression was normalized to gapdh mRNA. Data are presented as fold-change in gene expression in each group relative to control group. The primers of Asic1a, Asic1b, Asic2a, Asic2b, and Asic4 were adapted from previous study (Schuhmacher and Smith, 2016 (link)). Asic3 primers were designed based on Primer-BLAST (NCBI, NIH). The primer sequence for Asic3 were forward, 5′-TATGTGGCTCGGAAGTGCGGAT-3′, and reverse, 5′-CAGACACAAGTGTCCTTTCGCAG-3′.
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