Isolation of RNA and DNA From 1 g of frozen mucosal scrapings, total RNA (DNasefree) was isolated using TRIzol Ò reagent (Invitrogen) as described recently [34] . The yield per gram of tissue and the purity of the RNA were calculated from measurement of the extinction at 260 and 280 nm. The integrity of all RNA samples was checked by analyzing 5 lg of RNA on a denaturizing 1% (w/v) agarose gel. After ethidium bromide staining, the gel was scanned to calculate the 28S/18S peak ratio (volume 28S over volume 18S) for each preparation. RNA with a ratio [2 was considered of adequate quality to be used for real-time PCR and microarray analysis. A part of the isolated RNA was used to prepare RNA pools for microarray analysis. A control pool was prepared by mixing equal amounts of RNA isolated from the jejunum of the three uninfected piglets (n = 3). The same was done for the two infected piglets slaughtered at 12 h and for the two piglets slaughtered at 18 h. After gentle homogenization in lysis buffer, DNA was extracted from 0.5 g of frozen mucosal scrapings, and 4 lg of purified DNA was analyzed on a 0.9% agarose gel [22] .