Analysis of EMT markers by qRT–PCR

Total RNA was extracted using the RNeasy Mini kit (Qiagen, CA) with DNase I treatment and quality controlled by electrophoresis on 0.8% agarose gel. RT–PCR was performed on 0.5–1 μg total RNA with the SuperScript ViloTM cDNA Synthesis kit from Invitrogen. Gene expression was assessed by quantitative real-time PCR with the GeneAmp 7,500 system and Taqman chemistry (Applied Biosystems, CA). Each sample was tested in triplicate. The Δ-Ct method was used to calculate relative fold-changes normalized against three different housekeeping genes. Taqman Gene Expression Assay IDs (Applied Biosystems, CA) were: Mm00441533-g1 (Snail, snail1, {"type":"entrez-nucleotide","attrs":{"text":"NM_011427.2","term_id":"31981483","term_text":"NM_011427.2"}}NM_011427.2), Mm00441531-m1 (Slug, snail2, {"type":"entrez-nucleotide","attrs":{"text":"NM_011415.2","term_id":"42476308","term_text":"NM_011415.2"}}NM_011415.2), Mm00486906-m1 (E-cadh, cdh1, {"type":"entrez-nucleotide","attrs":{"text":"NM_009864.2","term_id":"118129809","term_text":"NM_009864.2"}}NM_009864.2), Mm00483213-m1 (N-cadh, cdh2, {"type":"entrez-nucleotide","attrs":{"text":"NM_007664.4","term_id":"161760626","term_text":"NM_007664.4"}}NM_007664.4), Mm00495564-m1 (zeb1, {"type":"entrez-nucleotide","attrs":{"text":"NM_011546.2","term_id":"118130126","term_text":"NM_011546.2"}}NM_011546.2), Mm99999915-g1 (GAPDH), Mm01197698-m1 (Gusb, {"type":"entrez-nucleotide","attrs":{"text":"NM_010368.1","term_id":"6754097","term_text":"NM_010368.1"}}NM_010368.1), Mm00607939-s1 (Actb, {"type":"entrez-nucleotide","attrs":{"text":"NM_007393.3","term_id":"145966868","term_text":"NM_007393.3"}}NM_007393.3).