Manual exfoliation plus immunomagnetic bead separation as an initial step toward translational research.
BACKGROUND
The development of biotechnologic platforms capable of high throughput analysis has ushered in a promising new era of translational medicine. However, most studies to date are based on in vitro cell lines or substitute models for human disease. Although these model systems have proven insightful, it is readily becoming apparent that human clinical tissue must be studied in order to fully understand all the nuances of human disease. Studies that are based on human tissue, however, are limited by qualitative and quantitative issues, factors often precluding their use in high throughput studies.


OBJECTIVE
To develop a simple and rapid tissue procurement protocol for use in obtaining a homogeneous epithelial cell population from clinical tissue and the recovery of nucleic acids and proteins of high quality and quantity. Also, to determine if the technique preserves tissue, thereby allowing morphologic correlation with molecular findings.


METHODS
Performance of manual exfoliation to procure cells from clinical resection specimens and use of immunomagnetic beads embedded with the antibody ber-Ep4 for the positive enrichment of a homogeneous epithelial cell population. Nucleic acids and proteins are then separated using a phenol plus guanidine thiocyante solution. Nucleic acids and proteins are quantitated and qualitatively analyzed using standard laboratory techniques.


RESULTS
Nucleic acids and proteins of high quality and quantity were recovered following manual exfoliation and immunomagnetic bead separation. Tissue architecture was not destroyed, thus permitting histologic and molecular correlation.


CONCLUSIONS
A simple and reproducible protocol is presented that may enable the molecular profiling of clinically resected tissue. Although the technique is currently limited to certain tissue and tumor types, further research will broaden its overall application.
Mojica
Wilfrido D
WD
Department of Pathology, University at Buffalo, State University of New York, Buffalo, USA. mojica@buffalo.edu
Arshad
Ayesha
A
Sharma
Sanjay
S
Brooks
Stephen P
SP
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002985:Clinical Protocols; D004273:DNA, Neoplasm / Q000032:analysis; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005260:Female; D023281:Genomics; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D008297:Male; D042282:Microdissection / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010865:Pilot Projects; D014176:Protein Biosynthesis; D040901:Proteomics; D012334:RNA, Neoplasm / Q000032:analysis; D013048:Specimen Handling / Q000379:methods*
D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012334:RNA, Neoplasm