A method for examining differential mRNA expression along the crypt-villus axis of the human small intestine.
1. The aim of this study was to devise a method of segregating crypt and villus cell subpopulations from endoscopic human small intestinal biopsies which might be used to examine changes associated with functional differentiation at the molecular level. 2. Routine endoscopic biopsies from the human small intestine were subjected to a modified protocol of mechanical disruption and chelation to yield subpopulations of different cell types. The purity and character of the cell populations isolated was assessed by measuring enzyme activity and thymidine incorporation and by histology. A guanidinium isothiocyanate method was adapted for small samples to extract RNA from the isolated subpopulations, and probes for RNA with a known predilection for crypt and villus cells were used to further investigate the application and usefulness of the technique. 3. Sequential histological examination during the segregation protocol demonstrated that different cell types were removed serially from the biopsy samples. Cell-type enrichment of the segregated subpopulations was demonstrated by differential alkaline phosphatase activity and by differences in thymidine incorporation in the samples isolated. Sufficient quantities of RNA could be extracted from the segregated subpopulations for Northern blot analysis and the differential expression of mRNA for sucrase-isomaltase and transferrin receptor was demonstrated in the villus and crypt subpopulations respectively. 4. Messenger RNA can be successfully extracted from different cell types segregated from routine human endoscopic small intestinal biopsies. This technique should prove useful for investigating the mechanisms regulating the functional differentiation of epithelial cells in the small intestine and the regulatory mechanisms governing absorption of macromolecules.
Department of Medicine, University of Liverpool, Liverpool L69 3BX, U.K.
E
E
Chua
Q
Q
Wang
P
P
O'toole
M
M
Lombard
D001706:Biopsy; D015152:Blotting, Northern; D002469:Cell Separation; D004386:Duodenum / Q000378:metabolism* / Q000473:pathology; D004415:Dyspepsia / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D011990:Receptors, Transferrin / Q000235:genetics; D013394:Sucrase-Isomaltase Complex / Q000235:genetics
D012333:RNA, Messenger; D011990:Receptors, Transferrin; D013394:Sucrase-Isomaltase Complex