Cel mir 238 3p

Total RNA was isolated from skin biopsy specimens, fibroblasts and endothelial cells using an miRNeasy mini kit (Qiagen). Serum miRNAs were isolated using a PAXgene blood miRNA system (Qiagen). An miScript reverse transcription kit (Qiagen) and miScript SYBR Green PCR kit (Qiagen) were used to measure the expression levels of selected miRNAs in a model 7500 real-time PCR system analyzer (Applied Biosystems). miRNA-specific primers and the miScript Universal Primer (Qiagen) were used. The expression of the U6B small nuclear RNA (RNU6B) was used as the endogenous control to normalize the sample data. The spiked Caenorhabditis elegans miRNA-238 (Qiagen, cel-miR- 238-3p) was used as an exogenous control to normalize the serum miRNA data. To detect mRNA, cDNA was prepared using a Reverse Transcription System (Promega, USA) and amplified using real-time PCR with SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara, Shiga, Japan) using the primers shown in Supplemental Table 1. GAPDH expression was used as the endogenous control to normalize the sample data. Relative expression levels were calculated using the 2−ΔΔCt method. The miRNA primers were purchased from RiboBio (China) and are listed in Supplemental Table 3.