Reverse transcriptase–PCR was performed using the Invitrogen Superscript III VILO kit according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on 50 pg cDNA in a Roche LightCycler 480 using Roche supermix for PCR (Roche, Boulogne-Billancourt, France). Polymerase chain reaction efficiency was determined for each primer set to calculate the expression ratio. For the stroma, normalization was performed using three housekeeping genes (vimentin, tyrosine 3 monooxygenase, β2-microglobulin), as previously described.
30 Primer sequences were BMP-1/mTLD (F: GTCTATGAAGCCATTTGCGG, R: GACGCTCAATCTCAAAGGAC), PCPE-1 (F: CTCAAACCAGGTGATCATGC, R: AGAGATGGGGCTAGGGGCCT), PCPE-2 (F: CGCCAGAGAGACCTGTTTTC, R: CCTCAGGAACTGTGATTTTC), TLL-1 (F: GGCTGGAGTTCTTACATCTACG, R: CTTATCTCCCCTCCACAAATCG), TLL-2 (F: GTATATGAAGCCATGTGTGG, R: GCCTTTCGATCTCGAAGGAC), COL1A1 (F: CGGCTCCTGCTCCTCTTAG, R: CTGTCCAGGGATGCCATCT), COL3A1 (F: GGCCCTCCTGGTATTCCTG, R: GCCAATTCCTCCTATGCCAG), vimentin (F: CAAGTCCAAGTTTGCTGACC, R: CTCCGGTACTCGTTTGACTC), tyrosine 3 mono-oxygenase (F: TGGATAAGAGTGAGCTGGTACA, R: CGTGTCCCTGCTCTGTTACG), β2-microglobulin (F: TTCTGGTGCTTGTCTCACTGA, R: CAGTATGTTCGGCTTCCCATTC). For the epithelium, we used three other housekeeping genes for normalization as previously described
29: ubiquitin C (F: AACCCACAGTATATCTTTGGCG, R: CCCTCACTAGGTTCGATGACTTC), tatabox (F: TGCCGAAAGATGCACAGATGA, R: TGTTGTCACATATCGGAAGGC), and β-actin (F: CGGTCCACCCGCCACCAGTTCGCCA, R: TCCCACCATCACACCCTGGTGCCTA). The fold change in gene expression was calculated using the 2 ΔΔCT ratio according to a previously described method.
31 All PCR products were checked by sequencing (Millegen, Toulouse, France).