Primers were designed as previously described [13 (link)]. Primers, synthesized by Eurogentec, were targeted against human sequences of integrin alpha1, alpha2, alpha3, alpha6, alpha7, alphaV, beta1, beta4, beta5, beta6, beta7, beta8, and cyclophilin A and beta-actin as reference genes (Table 1 ). Every set of primers has been designed using intron spanning assay as required by the Universal Probe Library Software from Roche Diagnosis. Non specific products formation has been verified for each set of primers with the melting curve realized by the LightCycler™ 480 Software.
Real-time pPCR was performed on the LightCycler™ 480 (Roche) using the LightCycler™ 480 SYBR Green 1 Master mix (Roche). The PCR reaction was performed in 20 μl volume containing 16 ng cDNA, 10 μl 2x LightCycler™ 480 SYBR Green 1 Master mix and 1 μl of primer mix (10 μM forward primer, 10 μM reverse primer). The PCR profile was as follows: 5 min at 95°C, followed by 45 cycles of 10 sec at 95°C, 10 sec at 60°C and 10 sec at 72°C.
The Ct value of each gene of interest was normalized to the Ct of the reference genes as follows : ΔCTnorm = Ctgoi-Ctref with Ctref = (CtbACT × CtCycloA)(1/2) with norm = normalized, goi = gene of interest, and ref = reference gene. ΔΔCT = ΔCT experimental condition - ΔCT control condition. Values were expressed as 2-ΔΔCt normalized using the 10% FCS condition as control.
Real-time pPCR was performed on the LightCycler™ 480 (Roche) using the LightCycler™ 480 SYBR Green 1 Master mix (Roche). The PCR reaction was performed in 20 μl volume containing 16 ng cDNA, 10 μl 2x LightCycler™ 480 SYBR Green 1 Master mix and 1 μl of primer mix (10 μM forward primer, 10 μM reverse primer). The PCR profile was as follows: 5 min at 95°C, followed by 45 cycles of 10 sec at 95°C, 10 sec at 60°C and 10 sec at 72°C.
The Ct value of each gene of interest was normalized to the Ct of the reference genes as follows : ΔCTnorm = Ctgoi-Ctref with Ctref = (CtbACT × CtCycloA)(1/2) with norm = normalized, goi = gene of interest, and ref = reference gene. ΔΔCT = ΔCT experimental condition - ΔCT control condition. Values were expressed as 2-ΔΔCt normalized using the 10% FCS condition as control.
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