Renal Glomerular and Tubular Specific Gene Expression Profiles from Laser Capture Microdissected Mouse Kidneys
Sennait Tzeggai, Jean Pearcey, Banu Sis. University of Alberta, Edmonton, Canada
Background: The kidneys serve several homeostatic functions including blood filtration and removal of wastes. The kidney is an anatomically complex organ composed of various distinct cell types and histological compartments. To gain understanding of molecular regulations in renal compartments, we established a method to isolate high-quality RNA from glomeruli and tubules of kidneys. We aimed at identifying glomerular-specific and tubular-specific genome-wide gene expression profiles.
Design: Three normal CBA mouse kidneys were cut at 7 µm thickness onto membrane slides using a cryostat and stained with H&E. We performed IR/UV laser capture microdissection to isolate glomeruli and cortical tubules from whole microscopic kidney sections. After microdissection, total RNA was extracted using the PicoPure RNA Isolation Kit. RNA quality and quantity were then measured using Agilent RNA 6000 Pico Kit and the Quant-iT RiboGreen method, respectively. Glomerular and tubular RNA samples were amplified with the RiboAmp HS PLUS Amplification Kit. Measurement of large-scale gene expression was done using Affymetrix mouse 430 2.0 microarrays.
Results: Pools of three replicates of 80 glomeruli and 80 tubules from 3 different kidneys yielded high RNA quality and quantity. We obtained a mean 79 µg and 51 µg of antisense RNA from microdissected glomeruli and tubules, respectively, after two amplification rounds of approximately 1 ng of input total RNA. Thus we obtained sufficient amounts antisense RNA to run Affymetrix microarrays. A class comparison between microdissected glomeruli and tubules showed a total of 5953 differentially expressed genes, of which 2878 genes with a prominent selective expression in the glomeruli. The glomerular and tubular selective gene lists included numerous novel genes in addition to genes with “a priori” known glomerular and tubular expression.
Conclusions: A systemic analysis of glomerular and tubular specific gene lists allows discovery of novel transcripts and pathways involved in molecular regulation of glomerular and tubular functions in the kidney.
Category: Kidney (does not include tumors)
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 261, Wednesday Afternoon