Sybr green sybr premix ex taq rt pcr kit

Total RNA from the fibroblasts were isolated using TRIzol RNA extraction protocol. The TaqMan® microRNA Reverse Transcription Kit (Applied Biosystems™) was used to measure the expression levels of miR-320a in a real-time PCR system analyzer (Bio-Rad IQ5), with a miR-320a specific primer. U6B small nuclear RNA (RNU6B) was used as endogenous control. cDNA was prepared by High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems®) and amplified by real-time PCR with SYBR Green (SYBR Premix Ex Taq RT-PCR kit; Takara, Shiga, Japan) and appropriate primers (listed below). Expression of the GAPDH was used as endogenous control to normalize the sample data. Relative expression levels were calculated with the 2^-ΔΔCt method. The following primers were used:

miR-320a—forward: 5′-CGAGCAAAAGCTGGGTTGA-3′; reverse: 5′-GTGCAGGGTCCGAGGT-3′

U6snRNA—forward: 5′-CTCGCTTCGGCAGCACATATACT-3′; reverse: 5′-ACGCTTCACGAATTTGCGTGTC-3′

As previously described (24 (link)), total RNA was isolated from mouse skin or lung tissue using Trizol (Invitrogen Life Technologies) according to the manufacturer's instructions. cDNA was prepared using the Reverse Transcription System (Promega). The expression of related genes was measured using gene-specific primers (shown in Table S1) with SYBR Green (SYBR Premix Ex Taq RT-PCR kit, Takara) and the 7500 real-time PCR system analyzer (Applied Biosystems). GAPDH expression was used as the endogenous control to normalize the sample data. Relative expression levels were calculated using the 2^−ΔΔCt method.

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.