Taqman primer probe assay

Total RNA was extracted from rat PFC by the phenol–guanidine isothiocyanate–chloroform method as previously described (56 (link)) and further purified using an RNeasy kit with on-column DNase digestion according to the manufacturer’s instructions (Qiagen, Manchester, UK). One microgram of total RNA was converted to complementary DNA using an AMV first-strand synthesis kit (Invitrogen) and c-fos expression quantified by quantitative reverse transcription polymerase chain reaction using a TaqMan primer-probe assay (no. Rn02396759_m1; Applied Biosystems, Warrington, UK) on an ABI Prism 7500 detection system (Applied Biosystems). Data were normalized to the β-actin housekeeping gene (assay ID 4352931E; Applied Biosystems), and relative expression was analyzed by the ΔΔCt method.

C57BL/6 arginase I (Arg1) and inducible nitric oxide synthase (iNOS) genes were accessed via GenBank accession no. NM_007482.3 and NM_010927.4, respectively. Based on the predicted TaqMan primer/probe assay (Applied Biosystems) locations given by the manufacturer, a fusion was synthesized with Arg1 bp 54 to 331, linked to iNOS bp 2648 to 2924. A NcoI site was added to the start of Arg1, and overlapping BamHI (GGATCC) and NcoI (CCATGG) sites were added to the end of NOS2. This fusion was created and cloned in pUC57 by GenScript. The lyophilized construct was reconstituted and transformed into TOP10 Escherichia coli for amplification. Cells were lysed, and amplified DNA was digested and gel purified. Total DNA was quantitated to calculate copy numbers appropriate for the qRT-PCR standard curve.

RNA was extracted from cells 24 hours after vehicle or SHH treatment using the RNeasy Plus Universal Mini Kit (Qiagen cat # 73404) following manufacturer's instructions. On column DNase treatment was included in the extraction protocol (Qiagen cat # 79254) to remove residual genomic DNA carryover. cDNA synthesis was carried out using iScript Reverse Transcription Supermix for RT-qPCR kit (Bio-Rad cat #170-8840) per manufacturer's instructions, and input RNA for cDNA synthesis was 100 ng. RT reaction was carried out as follows: 5 min 25°C, 30 min 42°C, and 5 min 85°C. For real-time PCR, TaqMan primer probe assays (Life Technologies; all FAM-labeled) for human GLI1, GLI2, GLI3, PTCH, SMO, SHH (cat # 4453320), SUFU (cat # 4448892), and β-actin (cat # 4333762) were used, and amounts of each assay were used according to manufacturer's recommendations. KiCqStart® Probe qPCR ReadyMix (Sigma cat # KCQS04) was used for gene expression analysis per manufacturer's instructions. Real-time reaction was run on a Bio-Rad CFX96 Touch PCR system as follows: 1 cycle 95°C 30 sec, 45 cycles 95°C 15 sec 60°C 30 sec. Data was normalized to β-actin using the 2^−ΔΔCt methodology and the fold change between vehicle and SHH-treated samples was plotted.