Sepasol rna 1 super g kit

RT-PCR analysis was performed to estimate the expression of Grpr mRNA in the Vc and cervical spinal dorsal horn of suncus (n=3 of each sex). Adult suncus were deeply anesthetized by intraperitoneal injection of 50 mg/kg body weight sodium pentobarbital and sacrificed by blood loss. Dissected tissues (the Vc and cervical spinal dorsal horn) from suncus were immediately fixed with RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80°C until RNA extraction. Total RNA was extracted from samples using the Sepasol-RNA I Super G kit (Nacalai Tesque, Kyoto, Japan) according to the manufacture’s protocol. The RNA concentration was measured using the Qubit RNA assay kit (Thermo Fisher Scientific). First-strand cDNA was synthesized from 100 ng of total RNA with oligo-dT primers using the Omniscript RT Kit (QIAGEN, Hilden, Germany) in a 20-μl reaction volume. The predicted suncus Grpr sequence (accession number in GenBank: suncus Grpr; LC149855) was obtained from an unpublished suncus genome resource. The sequences of primers for RT-PCR analysis were also designed based on this genome resource (see Table 2). The resultant PCR amplicons were electrophoresed on 2% agarose gels. Each RT-PCR study was repeated three times using independently extracted RNA samples from different animals.