Sybr green fast qpcr

RNA of adipose tissue was extracted using TRIzol reagent (Life Technologies, Grand Island, NY). 50 mg adipose tissue/sample was ground into homogenization in liquid nitrogen before RNA extraction. We employed the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany) to reverse mRNA into cDNA. For quantitative RT–PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the QuantStudio 5 Real-Time PCR System (Applied Biosystems, Foster City, USA). The sequence of primers are summarized in the Supplementary Table 1.

RNA isolation was performed using an RNeasy purification kit according to the manufacturer’s instructions (Quiagen, Hilden, Germany). For reverse transcription of RNA into DNA, the QuantiTect Reverse Transcription kit was employed (Quiagen, Hilden, Germany). For quantitative RT-PCR (qPCR), aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). RPL41 as HCC housekeeping gene and the average ΔC_T value of the HCC cell line 3sp were used to calculate the ΔΔC_T values for all cell lines. The ΔΔC_T values were used to graph the fold-change (RQ) via the formula RQ = 2^(−ΔΔC_T). Error bars show SE of ΔΔC_T, calculated with the following formula: SE(ΔΔC_T) = √((SE(ΔC_T Control)^2) ± (SE(ΔC_T Target)^2)). The primer sequences are: NRP2 (forward), 5’-CTGTGGGTCATCCGTGAGGAC-3’ and NRP2 (reverse) 5’-ATGGGTTCCATGCAGTTCTCCAG-3’; RPL41 (forward), 5’-CAAGTGGAGGAAGAAGCGA-3’ and RPL41 (reverse), 5’-TTACTTGGACCTCTGCCTC-3’.

Total RNA was extracted, treated with DNaseI and reverse transcribed using a RNA isolation and cDNA synthesis kit as recommended by the manufacturer (Quiagen, Hilden, Germany). Aliquots of cDNA were employed for Fast SYBR green qPCR (Applied Biosystems, Foster City, USA) 2 and quantified with the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). The following probes, forward and reverse primer sequences were used: CD32b: 5’- CCATCTGGACTGGAGCCAAC-3’ and 5’- TGGCTTGCTTTTCCCAATGC-3’; CD146: 5’-CGGGTGTGCCAGGAGAG-3’ and 5’-ACCAGTCCACTTGGCTGAAG-3’; vWF: 5’-TGGCAAGGTTTTTCAGGGGA-3’ and 5’-TAAGCAGGTGATGCAGAGGC-3’; Stab-1: 5’-ACGCTTCTAACGCCACCTTT-3’ and 5’-CCACACGATGACGTGGCTAA-3’; Stab-2: 5’-CACTATGTCGGGGATGGACG-3’ and 5’-GGGAGCGTAGGTGGAATACG-3’; LYVE-1: 5’-TACAGGACCCATGGCTGAGA-3’ and 5’-GGTGCCAAGCATTTCGGTTT-3’; RhoA: 5’-CCATCATCCTGGTTGGGAAT-3’ and 5’-CCATGTACCCAAAAGCGC-3’.