2.4. Real-Time Quantitative PCR

Total RNA samples from primary keratinocytes, derived from mouse back skin, were isolated. Following this, the cDNA was hybridized from 1 μg of the total RNA with a LeGene first strand cDNA synthesis system (LeGene Bioscience, San Diego, CA, USA) [31]. Several expression levels of mice keratin family (Krt6a, Krt5, and Krt14) were determined with a quantitative PCR test, as described in the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCt value compared to the normal mice sample was determined with StepOne software (Applied Biosystems). Gapdh was used as a house keeping gene and endogenous control. The sequence of the forward and reverse primer was 5′-CATGGCCTTCCGTGTTCCTA-3′ and 5′-GCGGCACGTCAGATCCA-3′ for the Gapdh gene, 5′-CCCTCTGAACCTGCAAATCG-3′ and 5′-GATCTGCTCCCTCTCCTCAGT-3′ for the Krt6a, 5′-TGCCCTGCCGTTTCTCTACT-3′ and 5′-TGATCTGCTCCCTCTCCTCA-3′ for the Krt5, and 5′-ACGAGAAGATGGCGGAGAAG-3′ and 5′-CTCTGTCTTGCTGAAGAACCATTC-3′ for the Krt14.