Mxpro 4

For each quantitative polymerase chain reaction (qPCR) reaction, 6.25 ng DNA was used, and for each sample, three technical replicates were performed. Primers for the nuclear ALB gene (located on chromosome 4) and for a 344‐bp fragment of the mitochondrial genome (nucleotides m.8151–8494), encompassing the MT‐TK gene were used: ALB exon 12 (GenBank NM_000477.6) forward: 5′‐AAT GCT GCA CAG AAT CCT TGGT‐3′, reverse: 5′‐TCA TCG ACT TCC AGA GCT GAAA‐3′; MT‐TK forward: 5′‐CGG GGG TAT ACT ACG GTC AA‐3′, reverse: 5′‐TTT TAT GGG CTT TGG TGA GG‐3′). Template DNA was amplified in the presence of each primer (0.5 µM) and reagents of the SensiMix SYBRHi‐ROX Kit (Bioline) in 12.5 µl‐reactions using the Stratagene Mx3005P qPCR system (Agilent Technologies). Carboxy‐X‐rhodamine (ROX) was used as passive reference dye. Cycling conditions: 10 min 95°C, 40 × [15 sec 95°C, 30 sec 60°C, 20 sec 72°C], followed by a dissociation segment for melting curve analysis. SYBR Green fluorescence data were analyzed using the MxPro 4.10 software (Agilent Technologies). The relative mitochondrial DNA (mtDNA) amount for each patient sample was obtained by normalization of the MT‐TK Ct values with the Ct values of the ALB gene. Ct values were calibrated to the DNA of peripheral blood from a healthy individual.

Cells were washed twice with PBS, and total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Equal amounts (1 μg) of RNA from each sample were used for cDNA synthesis using random primers and the M-MuLV reverse transcriptase enzyme (Thermo Scientific, Rockford, Illinois, USA). Quantification of the mRNA expression levels of WNT5A, IL-6 and IL-6R and the endogenous TATA box binding protein (TBP) in the samples was carried out on a Stratagene Mx3005P system (Agilent Technologies, Santa Clara, CA, USA) using Maxima Probe/ROX QPCR Master Mix (Thermo Scientific) and primers, TaqMan Gene Expression Assays Hs01075666_m1 and Hs00427620_m1, respectively (Thermo Fisher Scientific, Waltham, MA, USA). For the relative quantification of WNT5A, IL-6 and IL-6R levels, the comparative Ct method was performed using MxPro 4.10 software (Agilent Technologies, Santa Clara, CA, USA) and normalised against TBP.