Quantification of Desmoglein Genes by qPCR

SYBR Green qPCR was performed in a final volume of 22.5 μL, with 12.5 μL of SYBR Green PCR MIX (Promega, Madison, USA), 0.5 μL of each primer (10 μM), 6.5 μL of ultrapure water and 2.5 μL of cDNA (1:4) in the Rotor Gene-Q® thermocycler (Qiagen, Hilden, Germany). Primer sequences that amplify fragments of the Dsg1, Dsg2 and Dsg3 genes and of the human ribosomal gene (18S) as endogenous genes were used.13 (link), 14 (link), 15 (link) The primer sequences, the resulting amplified product (amplicon) and the amplification cycle parameters are detailed in Supplemental Table 1. The relative gene expressions of Dsg1, Dsg2 and Dsg3 were calculated using the equation 2 ^(−ΔcT), having the 18S rRNA as the reference gene; ΔcT was calculated by subtracting the cycle threshold (cT) value from the target gene (Dsg1, Dsg2 or Dsg3) from the cT value of the reference gene (18S).¹⁶ (link) To verify the specificity of each sequence, the obtained amplicon was purified (ExoSAP-IT®), sequenced (3500 Genetic Analyzer, Applied Biosystems, USA), and analyzed at the NCBI BLAST.