Stepone real time pcr systems software v2

Cortical tissue micro-dissection was carried out as previously described24 (link). In brief, dissected tissues were snap frozen in liquid nitrogen, lysed in trizol reagent at 4°C and RNA extracted using the RNeasy Micro extraction kit (Qiagen). Genomic DNA was digested before elution from column. Isolated RNA was then subjected to quantification and purity check (Nanodrop). Equivalent amounts of RNA were subjected to reverse transcription using a high capacity RNA-to-cDNA kit (Applied Biosystems) to obtain cDNA. qPCR reactions were set up using Taqman Universal Master Mix (Applied Biosystems) and Taqman probes used as follows: Aggrecan, Brevican, Neurocan, Versican, TenascinR, Hapln1, Hapln4, Mme, Adamts4, Adamts8, Adamts15, MMP15, MMP24, Has1, Has2, Has3, CSGalNAcT1, C6ST-1, Ptprz1, Reln, PTPσ, RTN4R, SST, Calb1, Calb2, and Gapdh. All probes were FAM-conjugated except Gapdh which was VIC-conjugated serving as internal control. PCR reactions were performed on a StepOnePlus™ Real-Time PCR system (Applied Biosystems) in 96-well plates using a standard curve protocol. To quantify gene expression, standard curves loaded with known amount of cDNA were used for each probe. All results were internally normalized to Gapdh expression, then calculated with reference to standard curves by the StepOne Real-Time PCR systems software v2.1 (Applied Biosystems).