Total RNA extraction and quantitative real-time PCR

Brain tissue samples were extracted from the glial scar region under a microscope (Leica, Solms, Germany). Total RNA was extracted from brain or cell samples using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA (1 µg) was reverse transcribed to cDNA using a Hifair® II First-strand cDNA Synthesis Kit (Yeasen Biotech, Shanghai, China). mRNA expression levels were quantified using a SYBR Green Master Mix (Exiqon, Vedbaek, Denmark), and Ct values for each sample and gene were normalized with respect to glyceraldehyde 3-phosphate dehydrogenase. The expression of miRNA was tested using a fast real-time PCR system (7900 HT, ABI, Foster City, CA) and the appropriate miRNA oligonucleotide primers (Qiagen, Hilden, Germany). The fold-change values were calculated by normalizing with respect to the control samples. PCR amplification was performed for 40 cycles, and the data were collected using SDS software (Applied Biosystems, Foster City, CA). The sequences of the mRNA oligonucleotide primers were used are listed in the table follows: