Reverse transcription and PCR

Total RNA was extracted with TRIzol (Invitrogen). The RNA was subjected to reverse transcription with random hexamer primer and Moloney murine leukemia virus reverse transcriptase (Promega). Real-time PCR was conducted in a LightCycler quantitative PCR apparatus (Stratagene) using the FastStart Universal SYBR Green Master (Rox). All the gene expression results are expressed as arbitrary units relative to expression of the gene encoding β-actin. The sequences of the primer pairs used are showed in Supplementary Table 1.

To detect the opioid receptors in OPCs and OLs, PCR was performed using diluted reverse transcription products, 35 cycles were used for DOR, KOR and MOR transcripts amplification, 27 cycles were used for PDGFRα, MBP and GAPDH transcripts amplification. All the products of RT-PCR were analysed by 1.5% agarose gel electrophoresis. The sequences of the primer pairs are showed in Supplementary Table 1.