Quantitation of gene expression by real time PCR
Cell lysates were collected 48h post-transfection in solution C (4M
guanidine thiocyanate, 25mM sodium citrate pH7, 0.1M β-mercaptoethanol).
Total RNA was extracted using phenol, chloroform as previously
described.(Spath et al., 2018) Total RNA (50ng) was reverse-transcribed
into cDNA by Superscript Reverse Transcriptase III (Thermo Fisher
Scientific). Expression of Tmem251 , Alpl and Col2a1was quantified by ABI Prism 7900 Fast Sequence Detector (Thermo Fisher
Scientific) by using specific primers (Table S5) and SYBR green (Thermo
Fisher Scientific). Data were normalized to 18S rRNA and relative
expression was calculated by using the formula: 2 -ΔCt*106, with ΔCt being target gene expression relative
to 18S ribosomal RNA.(Nilsson et al., 2007)