Premix sybr green 2

Real-Time PCR quantity method was used by Premix SYBR Green II (Qiagen, Germany) for measuring NT4, NT3, NGF, TrkA, TrkB, and TrkC mRNA expression levels (Applied Biosystems Step One, America). The reaction mixture was done in final volume in 20 microliters (includes 1 microliter of cDNA, 1 microliter of forward primer, 1 microliter of reverse primer, 7 microliters of DEPC water and 10 microliters of Syber Green) and each reaction in a duplicate form. Designing primers was done according to NT4, NT3, NGF, TrkA, TrkB, TrkC, and GAPDH genes in the NCBI gene bank and by a German company (Qiagen). Furthermore, GAPDH was used as the reference gene. The thermal program used in Real Time-PCR included: 95 °C for 10 min, 95 °C for 15 sec, and 60 °C for 1 min (40 cycle repetitions). Melt curve and standard curve were drawn and considered for evaluating data authenticity and optimization experiment conditions respectively and NT4, NT3, NGF, TrkA, TrkB and TrkC expression data were normalized using GAPDH (reference gene). Fold change of genes was measured by the R=2^-ctΔΔ formula (18 (link)).

Real-Time PCR quantity method was used by Premix SYBR Green II (Qiagen, Germany) for measuring CDK5 and GSK-3β mRNA expression levels (Applied Biosystems StepOne, America). Reaction mixture was done in final volume in 20 µL (includes 1 µL of cDNA, 1 µL of forward primer, 1 µL of reverse primer, 7 µL of DEPC water and 10 µL of Syber Green) and each reaction in duplicate. Designing of primers was done according to CDK5, GSK-3β and GAPDH genes in gene bank of NCBI and by German company, Qiagen. Usable primer sequences have been reported in Table 1. Furthermore, GAPDH was used as the reference gene. Thermal program used in Real Time-PCR included: 95°C for 10 minutes, 95°C for 15 s, 60°C for 1 min (40 cycle repetitions). Melt curve and standard curve were drawn and considered for evaluating data authenticity and optimization experiment conditions, respectively and CDK5 and GSK-3β expression data were normalized using GAPDH (reference gene). Fold change of genes was measured by Equation 2 (21 (link)).