Total RNA was isolated from biopsy samples and cells using a RNeasy Mini Kit (QIAGEN, Hilden, Germany), and DNA digestion was performed using the Turbo DNA-free Kit (Thermo Fischer Scientific, Ireland), following the manufacturers’ instructions. For RNA extraction from the cell line experiments, the triplicates of each condition were extracted and pooled for gene expression analysis. cDNA was synthesized using 1 μg total RNA using Reverse Transcriptase enzyme provided by Transcription First Strand cDNA Synthesis Kit (Roche, Ireland). PCR primers (Eurofins, Ireland) and probes (Roche) were designed using the Universal ProbeLibrary Assay Design Center (https://www.roche-applied-science.com/sis/rtpcr/upl/adc.jsp; Roche Applied Science, Indianapolis, IN, USA). PCR reactions were performed in duplicates/triplicates by using the LightCycler 480 system (Roche Applied Science). Positive and negative controls were also included. The genes analyzed included CEACAM1, CEACAM3, CEACAM5, CEACAM6, CEACAM7, CEACAM20, CCL20, and IL-8/CXCL8 (see Supplementary Table 3 for primer and probe sequences). β-actin was used as housekeeping gene. The 2−ΔΔ Ct method was used to calculate the relative changes in each of the genes relative to the expression of β-actin determined from real-time qRT-PCR experiments (28 (link), 29 (link)).
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