mRNA expression analysis by real-time PCR

Relative mRNA expression of the indicated genes in the indicated conditions was quantified using a 7500 Fast Real-Time PCR System (Applied Biosystems). To adjust for possible differences in the amount of template added to the reaction, 18s RNA served as an endogenous control (expression levels of the endogenous control were subtracted from expression levels of target genes). Each sample was assessed in replicate for both the target gene and the endogenous control. Each experiment was repeated at least three times to ensure statistical significance. Real-Time PCR primers (18 – 20 nucleotides) for the indicated genes were designed with Primer 3 http://frodo.wi.mit.edu/ according to stringent product size (75 – 150 nucleotides) and annealing temperature (58 – 63°C) specifications. Predicted unspecific primer binding and secondary structure formation were excluded using the BLAST and IDT tools http://eu.idtdna.com/Home/Home.asp