To determine the expression of FoxP3, TGF-β, IL-10, IL-17, and RORγT in MLNs, RT-qPCR was performed as described earlier (34 (link)). Briefly, total RNA isolated from the MLN cells was used to generate cDNA using cDNA synthesis kit from Qiagen (Germantown, MD, USA) and following the protocol of the company. SSO Advanced™ SYBR green PCR kit obtained from Bio-Rad (Hercules, CA, USA) was used to perform RT-qPCR on CFX96 RT-qPCR system (BioRad). The following primer sets (Primer Bank, Harvard Medical School) were used for RT-qPCR.
RT-qPCR was performed using the following PCR cycles (40 cycles) and under these conditions: initial activation step (15 min at 95°C), denaturing temperature (15 s at 94°C), annealing temperature (30 s at 60°C), and extension temperature and fluorescence data collection (30 s at 70°C) were used. Using NE ¼ 2_∆∆Ct, where Ct is the threshold cycle to detect fluorescence, normalized expression (NE) of mRNAs was calculated, and fold change of mRNA levels was normalized to β-actin (a housekeeping gene, ACTB).
RT-qPCR was performed using the following PCR cycles (40 cycles) and under these conditions: initial activation step (15 min at 95°C), denaturing temperature (15 s at 94°C), annealing temperature (30 s at 60°C), and extension temperature and fluorescence data collection (30 s at 70°C) were used. Using NE ¼ 2_∆∆Ct, where Ct is the threshold cycle to detect fluorescence, normalized expression (NE) of mRNAs was calculated, and fold change of mRNA levels was normalized to β-actin (a housekeeping gene, ACTB).
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