Superscript reverse transcription system

Quantitative real-time reverse transcription–polymerase chain reaction (qRT-PCR) was performed to evaluate the expression of the reference gene β-actin, two upregulated genes hsa_circRNA_101798 and hsa_circRNA_403250, and three downregulated genes hsa_circRNA_000957, hsa_circRNA_402437, and hsa_circRNA_091420 in three lesional and nonlesional skins of patients with vitiligo and three normal healthy skins so as to validate microarray data. The cDNAs were synthesized with 3 µg total RNA using specific primers by the Superscript Reverse Transcription System (Invitrogen, CA, USA). The qRT-PCR reaction system was in a total volume of 10 µL of the mixture, including 5 µL of 2 × master mix (Arraystar), 0.5 µL of 10 µM PCR forward primer, 0.5 µL of 10 µM PCR reverse primer, 2 µL of diluted first-strand cDNA, and 2 µL of ddH₂O. The primers of specific genes designed by Primer Premier 5.0 software are listed in Supplementary Table 1. The cycling program is 95°C for 10 min, followed by 40 amplification cycles at 95°C for 10 s and 60°C for 1 min (QuantStudio 5 Real-time PCR System, Applied Biosystems). The melt curve analysis was carried out after PCR to determine primer specificity, and the relative level of each circRNA was calculated using the standard curve method. The Student t test (two-tailed) was used for data analysis. P< 0.05 indicated a statistically significant difference.