Quant ittm 338 oligreen ssdna assay kit 339

Mouse brain homogenates at P1, 3, 5, 7, 12, and 16 were prepared (n = 5/age). Total RNA was isolated according to the manufacturer's instructions using the miRNeasy Micro Kit (Qiagen). cDNA was prepared using the QuantiTect Reverse Transcription Kit (Qiagen), and PCR was run on a LightCycler 480 (Roche, Sweden). The following primers were used: Olig2 QT01041089 and Spp1 QT00157724. Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimation of amplification efficiency, a standard curve was generated using a series of concentrations of cDNA. The amplified Spp1 transcripts were quantified with the relative standard curve and normalized to the concentration of total cDNA in each sample measured using the Quant‐iTTM 338 OliGreen ssDNA Assay Kit 339 (Invitrogen).

Oligodendrocytes from control‐, OPN‐KO‐, and OLs‐iOPN‐KI mice (n = 7) at P5 were isolated using MACS. Total RNA was isolated according to the manufacturer's instructions using the miRNeasy Micro Kit (Qiagen). cDNA was prepared using the QuantiTect Reverse Transcription Kit (Qiagen), and PCR was run on a LightCycler 480 (Roche, Sweden). The following primers were used: Mbp QT00198478, Plp1 QT00096096, Cnp QT00106764, and Mobp QT00154924. The amplified transcripts were quantified with the relative standard curve and normalized to the concentration of total cDNA in each sample measured using the Quant‐iTTM 338 OliGreen ssDNA Assay Kit 339 (Invitrogen).