Quantitative RT-PCR Analysis of ED-B FN mRNA

RNA extraction from AMt, NF, and Ty-82 was performed using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions. One µg of each RNA sample was reverse transcribed to cDNA by QuantiTect Reverse Transcription Kit (Qiagen GmbH), and 30-fold dilutions of cDNAs were analyzed by quantitative RT-PCR on iCycler-iQ5 Optical System (Bio-Rad Laboratories, Hercules, CA, USA), using SsoAdvanced SYBR Green SuperMix (Bio-Rad Laboratories, Hercules, CA, USA), running each sample in duplicate. Primers were designed from coding sequences published on Gene Bank database with the support of Beacon Designer v.7 Software (Premier Biosoft International, Palo Alto, CA, USA) (sequences are available upon request). Relative quantitative analysis was performed following 2^−ΔΔCt Livak method [24 (link)], and the geometric mean of four housekeeping genes (ACTB, ATP5B, GAPDH, HPRT) based on the GeNorm study [25 (link)] was used to normalize the ED-B FN mRNA expression.