Sso advanced sybr green pcr kit

To validate the expression of miRNAs and genes of interest, total RNAs isolated from pulmonary infiltrating mononuclear cells from the lungs of Naïve, OVA-veh, and OVA-res groups were used. The concentration and the quality of RNAs were analyzed using Nanodrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of RNAs were used for cDNA synthesis. To examine the expression of miRNAs, cDNAs were synthesized using miScript cDNA Synthesis kit and following the protocol of the company (Qiagen, Valencia, MD, USA). SYBR Green PCR kit from Qiagen was used and the protocol of the company was followed. To detect gene expression, SSO- Advanced SYBR Green PCR kit (Bio-Rad, Hercules, CA) was used and the protocol of the company was followed. Real-Time PCR was performed for 39 cycles and the details are as follows: 30 s 98°C (denaturation step), 60 s at 60°C (annealing step) and 60 s at 72°C (extension step, followed by incubation for 10 min at 72°C. The gene expression was normalized to GAPDH. GAPDH housekeeping gene was used because its expression is reliable for one kind of cells 41 (link), 42 (link). The PCR results of miRNAs expression were normalized to Snord 96A (small nucleolar RNA, C/D box 96A) was used as a control to assess the level of miRNA (43 (link)). The details of miRNAs and primer sequences for genes used in qPCR are described in Supplementary Table 1.

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).

To determine the expression of FoxP3, IL-17, IL-10, TGF-β, RORγT, and HMGB1 in DLN of the four groups, we performed RT-qPCR. In brief, cDNAs were generated using total RNAs isolated from the cells harvested from DLN of the four groups of mice. SSO Advanced™ SYBR green PCR kit from Bio-Rad (Hercules, CA, USA) was used. The following primers (Primer Bank, Harvard Medical School) were used for RT-qPCR (Table 1).
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 $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$4$}\right.$ 2_DDCt, 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). The Student's t-test was performed using GraphPad version 6.01 (GraphPad Software, INC., San Diego, CA) to determine significant differences in mRNAs level in the draining lymph nodes of all the four groups. Differences between treatment groups were considered significant when p < 0.05.