Ez 10 spin column total rna minipreps super kit

Human monocyte-derived macrophages were treated with P17 (200 µg/ml) and/or LTB4 (100 nM; Cayman Chemical Company) for 8 h.
The mRNA preparation was made using the EZ-10 Spin Column Total RNA Minipreps Super Kit (Bio Basic) using the manufacturer’s protocol. Synthesis of cDNA was performed according to the manufacturer’s recommendations (Thermo electron). RT-qPCR was performed on a LightCycler 480 system using LightCycler SYBR Green I Master (Roche Diagnostics). The primers (Eurogentec) were designed with the software Primer 3. GAPDH mRNA was used as the invariant control. Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. Primer sequences are listed in Table 1.

F4/80 colonic cells were isolated with the anti-F4/80 MicroBeads UltraPure, mouse (Miltenyi Biotec) as recommended by the manufacturer’s instructions. mRNA from F4/80+ cells, from mice colon, BMDM, and from IBD patients’ monocytes was extracted with the EZ-10 Spin Column Total RNA Minipreps Super kit (BioBasic) and reverse transcription of mRNA was performed with the Verso cDNA kit (Thermo Fisher Scientific) following the manufacturer’s recommendations.
RT-qPCR was performed on a LightCycler^® 480 system using LightCycler^® SYBR Green I Master (Roche Diagnostics). Mouse GAPDH and human 18S ribosomal mRNA were used as the invariant controls in gene expression. Serially diluted samples of pooled cDNA were used as calibration standards in each run for cDNA quantification. Primer sequences are listed in supplementary Table 2 and 3.

RNA was extracted from cells cultures using EZ‐10 Spin Column Total RNA Mini‐preps Super Kit (Bio Basic Inc) according to the manufacturer's instructions. RNA concentration was measured by NanoDrop1000 spectrophotometer (Thermo Scientific). cDNA was synthesized using Maxima First Strand cDNA Synthesis Kit (Thermo scientific), and PCR was performed using SYBR green and specific primers (Table 1) on a Light Cycler 480 (Roche). Expression levels were normalized to the house‐keeping gene GAPDH using Lightcycler advanced relative quantification programme (Roche).

Total RNA was extracted from both poPSCs cultured in the presence of steroids for 7 or 14 days and poPSCs cultured without addition of steroids. Total cellular RNA was isolated using the EZ-10 Spin Column Total RNA Mini Preps Super Kit (Bio Basic Canada Inc.; Markham, ON, Canada) according to the manufacturer’s protocol. The quantity and quality of the total RNA were ascertained by measuring the absorbance at 260 and 280 nm with a NanoDrop ND2000 Spectrophotometer (Thermo Fisher Scientific; Wilmington, DE, USA). Moreover, RNA samples were electrophoresed on a 1% (wt/vol) denaturing agarose gel to verify the RNA quality and stored frozen at −80 °C. First-strand cDNA was prepared by reverse transcription (RT) using 1 mg of total RNA, random primers, and a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA, USA) according to the manufacturer’s protocol. The 20-mL total reaction volume contained random primers, dNTP mix, RNAse inhibitor, and Multi Scribe Reverse Transcriptase. RT was performed in a Veriti Thermal Cycler (Applied Biosystems) according to the following thermal profile: (1) 25 °C for 10 min, (2) 37 °C for 120 min, and (3) 85 °C for 5 min. Genomic DNA amplification contamination was checked using control experiments, in which reverse transcriptase was omitted during the RT step. The samples were kept at −20 °C until further analysis.