2007 JASN IMPACT FACTOR 7.111	HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP
advanced	CURRENT ISSUE	ARCHIVES	JASN Express	ONLINE SUBMISSION

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by EIKMANS, M. Articles by BRUIJN, J. A. Search for Related Content PubMed PubMed Citation Articles by EIKMANS, M. Articles by BRUIJN, J. A.

Processing Renal Biopsies for Diagnostic mRNA Quantification

Improvement of RNA Extraction and StorageConditions

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Correspondence to Michael Eikmans, Leiden University Medical Center, Department of Pathology, P. O. Box 9600, Building 1, L1-Q, 2300 RC Leiden, The Netherlands. Phone: +31 71 5266625; Fax: +31 71 5248158; E-mail: MEikmans{at}Pat.AZL.NL

Abstract. The goal of this study was to improve a procedure for the extraction and storage of RNA from minute quantities of human renal tissue in clinical practice, using kidney biopsies and cadaveric donor kidneys unsuitable for transplantation. Collagen 1(IV) mRNA was analyzed as a measure for RNA integrity. The results show that at least 3 h may pass between microdissecting the renal tissue and the onset of cDNA synthesis without degradation of the glomerular mRNA. To extract the glomerular mRNA, microdissected glomeruli were incubated in a permeabilization solution. Treating glomeruli with collagenase IV before permeabilization had a deteriorating effect on the mRNA yield. The addition of reverse transcription mixture to the permeabilization solution in the presence of the glomeruli resulted in the highest cDNA yields. Storage of glomerular tissue in the presence of Nonidet P-40-based buffer for 1 wk at -70°C did not significantly affect the mRNA, but storage for 2 or 4 wk resulted in deterioration of the mRNA by approximately 40 and 95%, respectively. Furthermore, three methods for total RNA isolation from microdissected interstitial tissue were compared. An approximately 2.5 times higher yield of collagen 1(IV) mRNA was obtained with silica gel-based membrane spin technology than with a guanidine isothiocyanate/phenol chloroform or a lithium chloride/phenol chloroform method. Finally, this study shows for the first time reliable detection of collagen 1(IV) mRNA in biopsies that had been frozen for at least 10 yr at -70°C. These experiments have helped to improve a procedure for the processing of glomerular and interstitial tissue acquired from human kidney biopsies for mRNA analysis. This method is suitable for implementation in routine clinical practice.

This article has been cited by other articles:

				M. C. Roos-van Groningen, E. M. Scholten, P. M. Lelieveld, A. T. Rowshani, H. J. Baelde, I. M. Bajema, S. Florquin, F. J. Bemelman, E. de Heer, J. W. de Fijter, et al. Molecular Comparison of Calcineurin Inhibitor-Induced Fibrogenic Responses in Protocol Renal Transplant Biopsies J. Am. Soc. Nephrol., March 1, 2006; 17(3): 881 - 888. [Abstract] [Full Text] [PDF]

				M. Eikmans, H. J. Baelde, E. de Heer, and J. A. Bruijn Messenger RNA assessment in clinical nephrology: perspectives and progress of methodology Nephrol. Dial. Transplant., December 1, 2005; 20(12): 2598 - 2601. [Full Text] [PDF]

				M. Eikmans, M. C. Roos-van Groningen, Y. W.J. Sijpkens, J. Ehrchen, J. Roth, H. J. Baelde, I. M. Bajema, J. W. de Fijter, E. de Heer, and J. A. Bruijn Expression of Surfactant Protein-C, S100A8, S100A9, and B Cell Markers in Renal Allografts: Investigation of the Prognostic Value J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3771 - 3786. [Abstract] [Full Text] [PDF]

				K. Koop, M. Eikmans, H. J. Baelde, H. Kawachi, E. de Heer, L. C. Paul, and J. A. Bruijn Expression of Podocyte-Associated Molecules in Acquired Human Kidney Diseases J. Am. Soc. Nephrol., August 1, 2003; 14(8): 2063 - 2071. [Abstract] [Full Text] [PDF]

				M. Eikmans, H. J. Baelde, E. C. Hagen, L. C. Paul, P. H. C. Eilers, E. de Heer, and J. A. Bruijn Renal mRNA Levels as Prognostic Tools in Kidney Diseases J. Am. Soc. Nephrol., April 1, 2003; 14(4): 899 - 907. [Abstract] [Full Text] [PDF]

				D Barbaric, L Dalla-Pozza, and J A Byrne A reliable method for total RNA extraction from frozen human bone marrow samples taken at diagnosis of acute leukaemia J. Clin. Pathol., November 1, 2002; 55(11): 865 - 867. [Abstract] [Full Text] [PDF]

Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673