Quantitative SYBR Green RT-PCR Assay

One step SYBR Green real-time RT-PCR (Qiagen) was carried out in a LightCycler 480 II (Roche) according to manufacturer’s recommendations, as we previously reported [37 (link), 104 (link), 129 (link)]. Briefly, for standard curve preparation, 5-50 ng of total RNA were reverse transcribed using a SYBR Green mix (Qiagen) containing 0.5 μM primers. Agarose gel electrophoresis was used to visualize the size of the amplification products. cDNA purification was performed using the QIAquick Gel Extraction Kit (Qiagen). Serial dilutions of cDNA (20,000; 2000; 200; 20; 2; 0.2 fgs) were used for the absolute quantification of target gene expression. QuantiTect Primer Assays for KLF2, PPARγ, ARNTL, ZAP-70, Lck, PTPN13, RORC, MAP3K4, and SERPINB6 were purchased from Qiagen. The expression of each gene was normalized relative to the internal control 28S rRNA levels (forward 5′-CGAGATTCCTGTCCCCACTA-3′; reverse 5′-GGGGCCACCTCCTTATTCTA-3′, IDT). Melting curve analysis performed after real-time amplification revealed the uniformity of thermal dissociation profile for each amplification product. Samples without template or without reverse transcriptase were used as negative controls. Each RT-PCR reaction was performed in triplicate.