**2.4 Reverse transcriptase polymerase chain reaction**

The biopsy was snap frozen in liquid nitrogen. Frozen tissue was homogenised and total RNA was extracted according to the methods suggested by the manufacturer, following phenol-chloroform extraction and ethanol precipitation. Reverse transcription from 2.5 µg of total RNA was carried out using the SUPERSCRIPT™ First-Strand Synthesis System (Invitrogen, Lidingö, Sweden) with Oligo (dT) Primers (Life Technologies, Täby, Sweden). Resulting cDNA was stored at – 20C until use.

Lightcycler Q-PCR (Roche Diagnostics AB, Stockholm, Sweden) was performed using the FastStart DNA Master SYBR Green I (Roche Diagnostics AB, Stockholm, Sweden). PCR was performed containing 2 µl of each RT sample using the hot-start technique. MgCl2 concentration was optimised to 4 mM to obtain the highest signal intensity and lowest background. For each tested sample the copy number of the PCR products was calculated by dividing these values by the genometric mean copy number of the reference gene (GAPDH). The quantification was performed by the software supplied by Roche Diagnostics (Mannheim, Germany). The primer sequences, PCR products sizes and references are listed in Table 2.


IL; interleukin, GAPDH; Glyceraldehyde-3-phosphate dehydrogenase

Table 2. rt-PCR-related information

#### **2.5 Histology**

The fixed biopsy were dehydrated and embedded in paraffin. A histo-pathologist evaluation of mucosal inflammation and histological changes were performed in coded three-micron sections stained with eosin-hematoxylin. Histological evaluation of inflammation (number of mucosal lymphocytes, plasma cells and eosinophilic granulocytes) and morphometric investigations concerning: basal cell layer thickness (BCL), papillary length (PL), total epithelium thickness, and dilatation of intracellular spaces (DIS), were performed on the mucosal specimens.
