Abstract
Laser-capture microdissection and transcriptional profiling have enabled compartment- and cell-specific analysis of gene expression in chronic kidney disease, thus facilitating the investigation of pathophysiological associations between glomerular, tubular, and interstitial structures. Due to the pico- and nanogram amounts of RNA isolated from LCM-captured material linear RNA amplification protocols are necessary prior to real-time PCR and microarray analysis. In this chapter, we describe the isolation of renal tubule cells from cryocut sections from routine kidney biopsies, and the isolation and linear amplification of RNA for downstream purposes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Henger A, Kretzler M, Doran P et al (2004) Gene expression fingerprints in human tubulointerstitial inflammation and fibrosis as prognostic markers of disease progression. Kindey Int 65:904–917
Wenjun J, Eichinger F, Bitzer M et al (2009) Renal gene and protein expression signatures for prediction of kidney disease progression. Am J Pathol 174:2073–2085
Higgins J P, Wang L, Kambham N et al (2004) Gene expression in the normal adult human kidney assessed by complementary DNA microarray. Mol Biol Cell 15: 649–656
Rudnicki M, Perco P, Enrich j et al (2009) Hypoxia response and VEGF-A expression in human proximal tubular epithelial cells in stable and progressive renal disease. Lab Invest 89:337–346
Emmert-Buck MR, Bonner RF, Smith PD, et al (1996) Laser capture microdissection. Science 274:998–1001
Kohda Y, Murakami H, Moe OW et al (2000) Analysis of segmental renal gene expression by laser capture microdissection. Kidney Int 57:321–331
Nagasawa Y, Takenaka M, Matsuoka Y et al (2000) Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting. Kidney Int 57:717–723
Van Gelder RN, von Zastrow ME, Yool A et al (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA 87:1663–1667
Eberwine J, Yeh H, Miyashiro K et al (1992) Analysis of gene expression in single live neurons. Proc Natl Acad Sci USA 89:3010–3014
Rudnicki M, Eder S, Schratzberger G et al (2004) Reliability of t7-based mRNA linear amplification validated by gene expression analysis of human kidney cells using cDNA microarrays. Nephron Exp Nephrol 97:e86-e95
Noutsias M, Rohde M, Block A et al (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 14:3
Edgley AJ, Gow RM, Kelly DJ (2010) Laser-capture microdissection and pressure catapulting for the analysis of gene expression in the renal glomerulus. Methods Mol Biol 611:29–40
Woroniecki RP, Bottinger EP (2009) Laser capture microdissection of kidney tissue. Methods Mol Biol 466:73–82
Denning KM, Smyth PC, Cahill SF et al (2007) A molecular expression signature distinguishing follicular lesions in thyroid carcinoma using preamplification RT-PCR in archival samples. Mod Pathol 20:1095–1102
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Noppert, SJ., Eder, S., Rudnicki, M. (2011). Laser-Capture Microdissection of Renal Tubule Cells and Linear Amplification of RNA for Microarray Profiling and Real-Time PCR. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 755. Humana Press. https://doi.org/10.1007/978-1-61779-163-5_21
Download citation
DOI: https://doi.org/10.1007/978-1-61779-163-5_21
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-162-8
Online ISBN: 978-1-61779-163-5
eBook Packages: Springer Protocols