Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons

Klaile, Esther; Klassert, Tilman E; Scheffrahn, Inka; Müller, Mario M; Heinrich, Annina; Heyl, Kerstin A; Dienemann, Hendrik; Grünewald, Christiane; Bals, Robert; Singer, Bernhard B; Slevogt, Hortense

To analyze the gene expression of CEACAM1, CEACAM5, and CEACAM6, total RNA was extracted from 2×10 6 NHBE cells using the Qiagen RNeasy mini kit. Residual genomic DNA was removed by on-column incubation with DNaseI (Qiagen). A NanoDrop D-1000 Spectrophotometer (Thermo-Fisher Scientific) was then used to assess the amount and quality of the isolated RNA samples. First-strand complementary DNA (cDNA) was synthesized from 2 μg of RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). To detect the expression of the CEACAM genes by PCR, specific primers for each target were designed using the on-line primer-BLAST tool of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/ tools/primer-blast/). Possible secondary structures at the primer binding sites were taken into account by characterizing the nucleotide sequence of the regions of interest using the Mfold algorithm [44] . Primer design across different exon boundaries allowed for specific primer pairs targeting 7 isoforms of CEACAM1. Primers were also designed for CEACAM5, CEACAM6, two housekeeping genes, and IFNβ-1a.