Gene Expression Analysis of WSB1 and IL21R

To evaluate the expression levels of WSB1 and IL21R genes, RNA was isolated from PBL cultures using RNeasy Mini Kit (Qiagen, Hamburg, Germany) and 0.3 μg of total RNA from each sample was reverse transcribed into cDNA using 200 U of Superscript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) and 500 ng of Oligo (dT) (Life Technologies, Carlsbad, CA, USA), as described previously (36 (link)). Pre-synthesized Taqman® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) were used to amplify WSB1 (Hs00373204_m1), IL21R (Hs00222310_m1) and β-actin (Hs01060665_g1). cDNA was detected using QuantStudio 12K Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Each qRT-PCR assay was performed with 10 ng of cDNA in 10 μL of Taqman-PCR Master mix 2X (Applied Biosystems, Foster City, CA, USA) and 1 μL of primer/probe set and purified using deionized H₂O q.s. 20 μL. Gene expression was normalized to β-actin levels. Relative quantification was performed using the comparative threshold cycle (ΔΔCT) method (37 (link)–39 (link)).