Optimal method for RNA extraction from mouse glomeruli.
Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Due to their small size, mouse glomeruli cannot be isolated by relatively simple sieving techniques. Based on recently reported methods for the isolation of mouse glomeruli, we developed an RNA isolation technique by performing comparative methodological studies. Two standard RNA extraction methods were compared. In addition in separate experiments the influence was studied of protease inhibitors and freezing and thawing of whole kidney prior to glomeruli isolation, on the yield and degradation of RNA. Therefore kidneys were perfused with 10 ml 0.01 M PBS containing 1.25% Fe3O4 through the aorta. Kidneys were decapsulated and passed through a 75-microns metal screen. After pelletting and washing, tubes were placed against a magnet and pelleted glomeruli were washed three times. In a second experiment protease inhibitors were added to the PBS. As a third method, kidneys were frozen before the isolation of glomeruli. From isolated glomeruli RNA was extracted using either caesium chloride or lithium chloride method. The yields of RNA (OD 260) were highest using the lithium chloride method. Hybridization of Northern blots of extracted RNA with cDNA probes showed the best results when RNA was extracted using the lithium chloride method, while the caesium chloride method led to considerable degradation of RNA. Freezing of kidney tissue prior to RNA extraction led to the virtual absence of any signal. We then applied this method successfully in an in-vivo model of experimental lupus nephritis. This is the first description of an optimal protocol for the extraction of RNA from mouse glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)
Baelde
J J
JJ
Department of Pathology, University of Leiden, The Netherlands.
Bergijk
E C
EC
Hoedemaeker
P J
PJ
de Heer
E
E
Bruijn
J A
JA
D000818:Animals; D002586:Cesium; D002712:Chlorides; D003094:Collagen / Q000235:genetics; D018076:DNA, Complementary; D005069:Evaluation Studies as Topic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007678:Kidney Glomerulus / Q000737:chemistry*; D018021:Lithium Chloride; D008181:Lupus Nephritis / Q000235:genetics / Q000378:metabolism; D008297:Male; D008722:Methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*
D002712:Chlorides; D018076:DNA, Complementary; D002586:Cesium; D012313:RNA; D003094:Collagen; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D018021:Lithium Chloride; C028019:cesium chloride