Direct-zol RNA Kits

Protocol tips

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- Highly effective when processing various sample types in TRI Reagent®, TRIzol®.

Upstream tips
- Highly effective when processing various sample types in TRI Reagent®, TRIzol®.

Publication protocol

After 48 h of activation, cells were detached with ice-cold 2.5 mM EDTA in PBS, washed once with PBS, pelleted and lysed in Trizol (Sigma-Aldrich, St. Louis, MO). RNA was isolated using Direct-zol RNA MiniPrep column system (Zymo Research, Irvine, CA) including a DNase I digestion step (15 min, RT). The RNA quantity was measured by NanoDrop (Implen). In addition, 10% of randomly selected RNA samples were tested in the Agilent TapeStation (Agilent Technologies) to check RNA integrity number (RIN) (all samples RIN > 8.5) and to crosscheck the concentration with those measured by NanoDrop. The cDNA synthesis was performed according to the manufacturer's recommendations using 200 ng RNA for DH82, U937 and human MDMs and 100 ng for canine MDMs using iScript cDNA Synthesis Kit (BioRad, Hercules, CA). For each donor, RNA from all conditions was pooled and used for the reverse transcriptase negative control (RT-). RT-qPCR was performed on a QuantStudio™12K Flex system (Applied Biosystems, 45 cycles; Tm 59–61 °C; 96 well plate and plate cover (Applied Biosystems) using a Solis Biodyne Supermix (Solis Biodyne, Tartu, Estonia). Target genes were selected according to human literature, concerning a strong discrimination between M1 and M2a macrophages (Martinez et al., 2006, Röszer, 2015). The primers (Supplementary Table S1) were either selected based on the recent literature or designed using the NCBI Blast and the Oligo Analyzer Tool (Thornton and Basu, 2011). Every plate included pooled RT-per donor and NTC per gene controls. M0 for DH82 and U937 M1 and M2a, GM-CSF for M1 MDMs and M-CSF for M2a MDMs were used as controls. A standard curve was generated and amplification efficiency (E) was calculated from the slope s of this curve (E = 10(− 1/s) − 1) using the QuantStudio 12k Flex Software v1.2.3 (Applied Biosystems). Quantification cycle (Cq) values were corrected by the term Cq × log10 (E + 1)/log10(2) (Robledo et al., 2014). For evaluating reference genes in canine macrophages, we tested ten candidates and chose ornithine decarboxylase antizyme (OAZ1) based on a stable expression profile over all activation conditions (data not shown). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference gene for human cells based on Martinez et al. (2006). After normalizing the data with the reference gene (OAZ1 for DH82 and canine MDMs; GAPDH for human MDMs and U937). ΔΔCq levels were calculated and presented as log2 fold change for M2a versus M1 macrophages. The mean fold change expression was calculated for M1/M2a versus M0 in DH82 and U937 and M1 versus GM-CSF, M2a versus M-CSF for MDMs, in case of a negative value the reciprocal value is presented.

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