Biorad cfx connect real time system

Real-time PCR was performed as previously described^{22 (link)}. Briefly, RNA was extracted from whole tissue by RNeasy mini kit (Qiagen, Venlo, Netherlands). Complementary DNA was reverse transcribed from 1 µg total RNA with Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific) using random hexamer oligonucleotides for priming (Thermo Fisher Scientific). The amplification was performed with a Biorad CFX-Connect Real-time-System (Thermo Fisher Scientific) using the SYBR Green (Eurogentec, Seraing, Belgium) or TaqMan (Thermo Fisher Scientific) detection system. Data were analyzed using the Bio-Rad CFX Manager 3.1 software. The mRNA content for Cxcl12, Ccl2, Cxcl9, and Ccl21 was normalized to the hypoxanthine–guanine phosphoribosyltransferase (Hprt) mRNA for mouse genes. Gene expression was quantified using the ∆∆Ct method. The expression level was arbitrarily set to 1 for one sample from the PBS group and the values for the other samples were calculated relatively to this reference. The sequence of primers is in Supplementary Table 6.

Real-time PCR was performed as previously described (51 (link)). Briefly, RNA was extracted from whole tissue by RNeasy mini kit (Qiagen, Venlo, Netherlands). Complementary DNA was reverse transcribed from 1 µg total RNA with Moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific) using random hexamer oligonucleotides for priming (Thermo Fisher Scientific). The amplification was performed with a Biorad CFX-Connect Real-time-System (Thermo Fisher Scientific) using the SYBR Green (Eurogentec, Seraing, Belgium) or TaqMan (Thermo Fisher Scientific) detection system. Data were analyzed using the software supplied with the Sequence Detector (Life Technologies). The mRNA content was normalized to the hypoxanthine-guanine phosphoribosyltransferase (Hprt) mRNA for mouse genes. Gene expression was quantified using the ΔΔCt method.