Total RNA extraction was performed using either the TRIzol ® reagent (Invitrogen Life Technologies, Karlsruhe, Germany) or the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufactures' protocols. One µg of total RNA was used for cDNA synthesis by reverse transcription (RT) in a total volume of 20 µl using either the Reverse Transcription System ® (Promega, Mannheim, Germany) or the First-Strand cDNA Synthesis ® Kit (MBI Fermentas, St. Leon-Rot, Germany). The RT product (1 µl) was amplified by polymerase chain reaction (PCR) with Taq polymerase (Qiagen). After an initial denaturation step at 95°C for 45 s, 35 PCR cycles (denaturation: 95°C, 30 s; annealing: 60°C, 1 min; extension: 72°C, 1.5 min) and a final extension step at 72°C for 15 min were performed. The primers were designed using the Primer3 software. Gene-specific primers were selected that exhibited > 2 mismatches at the 3'-end of the oligonucleotide to all other members of the canine CEACAM family, except for CEACAM28 and CEACAM30 (see below). The primers were validated by eye using multiple nucleotide sequence alignments of the aforementioned CEA family members. The primer sequence and the predicted sizes of the amplified products are summarized in Table 3 . Canine GAPDH cDNA was amplified using GAPDH.dogforward 5'-GCCAAAAGGGTCATCATCTC and GAPDH.dog reverse 5'-GCCCATCCACAGTCTTCT primers at a annealing temperature of 56°C and 30 cycles. Eight µl of each PCR were analyzed by electrophoresis on a 1.8 % agarose gel and visualized by ethidium bromide staining. The amount of cDNA was quantified by endpoint determination with the Quantity 1 ® software (Bio-Rad Laboratories, Munich, Germany).