Visualizar Tese - Instituto de Biociências - Unesp

Andreo Fernando Aguiargenerated by 7300 System SDS software (Life Technologies, Carlsbad, CA, USA) foreach gene analyzed. The analysis of all standard curves showed high linearity(r2=0.99). PCR efficiency (Ex) was calculated from the equation Ex = 10 -1 -1/slope-1. Slope of -3.32 implies in a reaction efficiency of 100%. All genes showed slopearound -3.32 and estimates of efficiency were between 99.5% and 100.5%. Primers forall genes were obtained using Primer3 software, available at webpagehttp://frodo.wi.mit.edu/primer3/, from published sequences in GenBank(www.pubmed.com) and synthesized by Life Technologies (Carlsbad, CA, USA) (Table1). Dissociation curves and agarose gel electrophoresis were performed to confirm theamplification of only one target sequence for each primer. Reaction controls were madewithout cDNA template to investigate possible contamination of reagents. Geneexpression was compared between individual samples using the ∆∆Cq methoddescribed by Livak and Schmittgen (36). It was also necessary to perform the correctionof experimental variability between different samples, for example, amount of RNA andreverse transcription reaction efficiency, prior to the final quantification. Datanormalization for at least three reference genes is the most accepted method to avoidsuch disparities (59). The choice of appropriate genes is crucial for reliable results, andthe expression level of these genes must remain unchanged for different experimentalconditions (59). Expression level of three genes, glyceraldehydes-3-phosphatedehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT)and TATA box binding protein (TBP) (Table 1), was assessed through DataAssistsoftware (Life Technologies, Carlsbad, CA, USA) and then used as reference genes fordata results normalization.51