Processing renal biopsies for diagnostic mRNA quantification: improvement of RNA extraction and storage conditions.
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 alpha1(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 degrees 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 alpha1(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 alpha1(IV) mRNA in biopsies that had been frozen for at least 10 yr at -70 degrees 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.
Eikmans
Michael
M
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Baelde
Hans J
HJ
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
DE Heer
Emile
E
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Bruijn
Jan Anthonie
JA
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
D001706:Biopsy; D002102:Cadaver; D003094:Collagen / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D004210:Dissection; D006801:Humans; D007678:Kidney Glomerulus / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014021:Tissue Preservation / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D003094:Collagen