Total mRNA was prepared using the RNeasy Mini Kit and reverse-transcribed with the QuantiTect Reverse Transcription Kit (both from QIAGEN, Hilden, Germany). Complementary DNA was amplified using SYBR Green PCR (polymerase chain reaction) Master Mix (QIAGEN) and gene-specific primers in a LightCycler 480 device (Roche, Mannheim, Germany). Normalization across samples was performed using geometric average of constitutive gene expression of β-glucuronidase. Gene expression levels were calculated according to the equation 2−ΔΔCT, where ΔCT was defined as CTgene of interest − CTendogenous control and ΔΔCT was defined as ΔCTgene of interest − ΔCTreference. ΔCT values were used for statistical comparison. The following primers were used:
Ddr1, 5′-TCCATAGACCAGAGGGATC-3′ (forward) and 5′-CAGGGCATAGCGGCACTTGG-3′ (reverse);
E-cadherin, 5′-CAGGTCTCCTCATGGCTTTGC-3′ (forward) and 5′-CTTCCGAAAAGAAGGCTGTCC-3′ (backward);
Epcam, 5′-CAGTGTACTTCCTATGGTACACAGAATACT-3′ (forward) and 5′-CTAGGCATTAAGCTCTCTGTGGATCTCACC-3′ (backward);
Erbb-2, 5′-TCCCCAGGGAGTATGTGAGG-3′ (forward) and 5′-GAGGCGGGACACATATGGAG-3′ (backward)
Erbb-3, 5′-GCCCAATCCTAACCAGTGCT-3′ (forward) and 5′-AGCCTGTAATCTCCCGGACT-3′ (backward);
Grlh2, 5′-CACCTCTCAAGACTGTTACAAGACT-3′ (forward) and 5′-CGAGATGAGTGGACTTGCTATCTC-3′ (backward);
Gusp, 5′-CAACCTCTGGTGGCCTTACC-3′ (forward) and 5′-GGGTGTAGTAGTCAGTCACA-3′ (backward);
Krt19, 5′-CTACCTTGCTCGGATTGAGGAG-3′ (forward) and 5′-AGTCTCGCTGGTAGCTCAGATG-3′ (backward);
N-cadherin, 5′-AGGGTGGACGTCATTGTAGC-3′ (forward) and 5′-CTGTTGGGGTCTGTC-3′ (backward);
Rab25, 5′-TGAGCCAAGATGGGGAATCG-3′ (forward) and 5′-GGAGAACTCAACCCCGATGG-3′ (backward);
Slug, 5′-TCCCATTAGTGACGAAGA-3′ (forward) and 5′-CCCAGGCTCACATATTCC-3′ (backward);
Snail, 5′-GCGGAAGATCTTCAACTGCAAATATTGTAAC-3′ (forward) and 5′-GCAGTGGGAGCAGGAGAATGGCTTCTCAC-3′ (backward);
Twist, 5′-CGGGTCATGGCTAACGTG-3′ (forward) and 5′-CAGCTTGCCATCTTGGAGTC-3′ (backward);
Vimentin, 5′-CGGAAAGTGGAATCCTTGCA-3′ (forward) and 5′-CACATCGATCTGGACATGCTGT-3′ (backward);
Zeb1, 5′-CCATACGAATGCCCGAACT-3′ (forward) and 5′-ACAACGGCTTGCACCACA-3′ (backward);
Zeb2, 5′-CCGTTGGACCTGTCATTACC-3′ (forward) and 5′-GACGATGAAGAAACACTGTTGTG-3′ (backward).
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