Sybr green real time rt pcr

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.

Total RNA was isolated using RNeasy kit (Qiagen). The quality (260/280 ratio) and quantity of RNA collected were measured by a Pearl nanophotometer (Implen, Munich, Germany). One step SYBR Green real-time RT-PCR (Qiagen) was carried out in a LightCycler 480 II (Roche) according to the manufacturer’s recommendations. The quantification of RORC mRNA relative to the 28S rRNA levels was performed as we previously described [21 (link)]. Each RT-PCR reaction was performed in triplicates.

Total RNA was isolated using the RNeasy Kit (Qiagen) and quantified using the
Pearl nanophotometer (Implen). One step SYBR Green real-time RT-PCR (Qiagen) was
carried out in a Light-Cycler 480 II (Roche) according to the
manufacturer's recommendations, as we previously reported [17 (link), 18 (link)]. QuantiTect Primer Assays were purchased from Qiagen. The
expression of each gene was normalized relative to 28S rRNA levels.
Amplifications were performed in triplicate on 70 ng RNA/test for target genes
and 2 ng RNA/test for 28S rRNA.