Real mastermix sybr green detection system

Total RNA was extracted with NucleoSpin RNA/protein (Macherey-Nagel, Duren, Germany) and quantified by NanoDrop (Thermo Scientific, Wilmington, USA), which also evaluates RNA integrity.
The qRT-PCR was performed on an Eppendorf Realplex 4 Mastercycler using the Real MasterMix SYBR Green detection system (Eppendorf, Milan, Italy) in a total volume of 10 μl loaded in triplicate. Primers for fibroblast and myofibroblast markers, fibroblast-specific protein-1 (S100A4, NM_002961) and αSMA (NM_001613), as well as COL1 (NM_000088), FN (NM_002026), CXCR4 (NM_00100854), and β-actin (NM_001101, housekeeping gene) were supplied by Primerdesign (Primerdesign, UK).
The melting curve was performed to confirm the specificity of the SYBR green assay. The qRT-PCR was performed on each independent in vitro experiment on cultures of fibrocytes isolated from all the enrolled SSc patients and HSs. Gene expression values were calculated using the comparative ΔΔCT method [18 (link)].

Total RNA was obtained from cultured human SSc skin fibroblasts using NucleoSpin RNA/protein (Macherey-Nagel) and quantified by nanodrop, which was also used to evaluate the RNA integrity, in accordance with the manufacturer’s protocol (Thermo Scientific, Wilmington, USA). For each experimental condition, first­strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription Kit (Qiagen, Milan, Italy).
The qRT-PCR was performed on an Eppendorf Realplex 4 Mastercycler using Real MasterMix SYBR Green detection system (Eppendorf, Milan, Italy) in a total volume of 10 μL loaded in triplicate. Primers for α-SMA (NM_001613), S100A4 (NM_002961), COL-1 (NM_000088), FN (NM_002026), and β­actin (NM_001101, housekeeping gene) were supplied by Primerdesign (Primerdesign, UK).
The gene expression values were calculated using the comparative ΔΔ cycle threshold (ΔΔCT) method and corresponded to the expression level (fold increase) of the target gene compared to untreated cells, taken as the unit value [27 (link)]. In all qRT-PCR assays, the melting curve was performed to confirm the specificity of the SYBR green assay, and the qRT-PCR was performed on six independent experiments on cultured human SSc skin fibroblasts.

Fibrocytes were cultured for 8 days whereas skin fibroblasts were cultured up to 80% confluency prior to treatment with CTLA4-Ig, as described in the “Cell culture and treatments” section above.
Total RNA was extracted with NucleoSpin RNA/protein (Macherey-Nagel) and quantified by NanoDrop (Thermo Scientific), which also evaluates RNA integrity, in accordance with the manufacturer’s instructions. For each experimental condition, first-strand cDNA was synthesized from 1 μg total RNA using the QuantiTect Reverse Transcription Kit (Qiagen).
qRT-PCR was performed on an Eppendorf Realplex 4 Mastercycler using the Real MasterMix SYBR Green detection system (Eppendorf) in a total volume of 10 μl loaded in triplicate. Primers for CD86 (NM_175862.4), COL I (NM_000088), FN (NM_002026), TGFβ (NM_000660), αSMA (NM_001613), S100A4 (NM_002961), CXCR2 (NM_00116829), CXCR4 (NM_00100854), CD11a (NM_00111438), and β-actin (NM_001101, housekeeping gene) were supplied by Primerdesign.
Gene expression values were calculated using the comparative ΔΔCT method and they corresponded to the expression level (fold-increase) of the target gene compared with the calibrator sample (untreated cells) taken as the unit value by definition [32 (link)]. In each qRT-PCR assay, the melting curve was performed to confirm the specificity of the SYBR green assay.