Quantifying RANKL and NR3C1 Expression

Expression of RANKL was analyzed using qPCR with RNA samples obtained in gain-of-function and loss-of-function experiments. The RNA was extracted from cells and the complementary DNAs (cDNAs) were synthesized using High-Capacity cDNA Reverse Transcription kits (Thermo Fisher Scientific, Waltham, MA, USA), with gene expression analyses performed as described below. Expressions of RANKL, NR3C1 were also analyzed using qPCR assays [12 (link)]. For qPCR, 5× Hot FirePol EvaGreen qPCR Mix Plus (Solis, BioDyne, Tartu, Estonia) was used, following the manufacturer recommendations, on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany). Concentration of each primer in the qPCR reaction was 150 nM. Nucleotide sequences of primers are listed in Supplementary Table S1. All of the cDNA samples were diluted to the final concentration of 2.5 ng/µL. All of the samples were quantified in triplicate. Dilution series of cDNAs were prepared to create a relative standard curve, and absolute quantification of the data was performed using the second derivative maximum method (LightCycler 480, Software version 1.5; Roche Diagnostics, Mannheim, Germany). All of the data were normalized to the internal housekeeping genes of ribosomal protein, large, P0 (RPLP0) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).