Comparison of different methods to obtain and store liver biopsies for molecular and histological research

Abstract

Comp Hepatol. 2009 Jul 8;8:3. Comparison of different methods to obtain and store liver biopsies for molecular and histological research. Hoffmann G, Ijzer J, Brinkhof B, Schotanus BA, van den Ingh TS, Penning LC, Rothuizen J. Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, University Utrecht, Yalelaan 104, 3584 CM Utrecht, the Netherlands. L.C.Penning@uu.nl. ABSTRACT: BACKGROUND: To minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1. RESULTS: RNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1-4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies. CONCLUSION: At least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field. PMID: 19586524 [PubMed - in process]