Light cycler 2.0 real time pcr

The total RNA was extracted from the pituitary tissue of cultured cells using RNeasy Plus Mini Kit purchased from Qiagen (Valencia, Ca). The amount of RNA was estimated using a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE) and reverse transcribed with Transcriptor First Stand cDNA Synthesis Kit obtained from Roche Applied Science (Indianapolis, IN). An analysis of the relative gene expression was performed using quantitative real-time PCR and the comparative Ct method [29 (link), 30 (link)]. For this, the LightCycler TaqMan Master mix and Lightcycler 2.0 Real-time PCR (Roche Applied Science, Indianapolis, IN) system were used. To compare the relative expression levels of the transcripts, the levels were calibrated against Gapdh and shown as percentage values with Gapdh expressed as 100%. Applied Biosystems predesigned Taq-Man Gene Expression Assays were used for Gapdh Mm99999915_g1 and Gnrhr Mm00439143_m1. The target gene expression levels were determined by comparative 2^(− δC(T)) quantification method using GAPDH as the reference gene. Linear regression analysis with mean amplification C(T) values showed no effects of age, gender or GnRH treatment on the expression of GAPDH mRNA in the anterior pituitary tissue and cells. This justified the use of GAPDH as a reference gene for analysis of mRNA expression.