Epithelial cell preparation for proteomic and transcriptomic analysis in human pancreatic tissue.
Standardized sample preparation procedures constitute a prerequisite for obtaining reliable and reproducible results in gene expression research in humans. In particular, in diseases such as pancreatic cancer and pancreatitis, isolating epithelial cells is an important step preceding such research. In pancreatic tissue, the high amount of RNAases is a further problem when it comes to obtaining high-quality RNA, and the presence of secreted proteases accelerates protein degradation. We developed a successful method that addresses these different problems. This method, which uses epithelial cell surface antibody Ber-Ep4, proteases, and RNAases inhibitors, leads to a significant enrichment (> 95% purity) of epithelial cells from fresh human tissue samples and allows for both proteomics (Western Blot, 2D PAGE) and transcriptomics studies (rtPCR, cDNA microarray). Compared with other cell purification procedures, this method is characterized by several advantages: a large quantity of cells available for downstream analysis, combined transcriptomics and proteomics studies using the same samples, better reproducibility of proteomics studies, and an acceptable yield (63%) for gene expression arrays studies. Moreover, a quality control protocol addressing the needs of the industry and the requirements of regulatory agencies is proposed.
Kellner
Udo
U
Department of Pathology & Cytology, Klinikum Minden, Friedrichstrasse 17, 32427 Minden, Germany. udo.kellner@klinikum-minden.de
Steinert
Ralph
R
Seibert
Volker
V
Heim
Steffen
S
Kellner
Angela
A
Schulz
Hans-Ulrich
HU
Roessner
Albert
A
Krüger
Sabine
S
Reymond
Marc
M
D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010179:Pancreas / Q000378:metabolism* / Q000473:pathology; D010182:Pancreatic Diseases / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D040901:Proteomics; D011786:Quality Control; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods* / Q000592:standards; D014158:Transcription, Genetic
D012333:RNA, Messenger