Improvement of extraction and processing of RNA from renal biopsies.
BACKGROUND
Assessment of mRNA levels has the potential to predict renal outcome. The objective of this study was to optimize several steps in the protocol for obtaining cDNA from routine clinical kidney biopsy material.


METHODS
RNA integrity was compared between different methods for extraction of RNA, synthesis of cDNA, and storage of renal tissue. Hereby, RNAlater, an RNA-preserving compound, was tested for implementation in a protocol for renal biopsies that combines routine histology and RNA expression studies. Gel electrophoresis and real-time polymerase chain reactions (PCR) were outcome parameters for assessment of RNA integrity.


RESULTS
The Trizol method rendered higher RNA yields from fresh renal tissue than the NP40 method and RNeasy spin columns. RNA yields were not affected when renal tissue was stored at -70 degrees C for up to 2 months in phosphate-buffered saline (PBS). cDNA levels obtained using avian myeloblastosis virus (AMV) reverse transcriptase (RT) were at least twice as high as those obtained with Sensiscript and Superscript RT. RNAlater maintained RNA integrity in whole renal cortex stored at 4 degrees C for up to 3 months. Dissection of small biopsies in RNAlater rendered similar RNA yields in comparison with dissection in PBS, but the yield of glomeruli from the cortices was 50% lower (P < 0.005). Integrity of RNAlater-treated tissue, evaluated by light microscopy and immunofluorescence, was diminished.


CONCLUSIONS
This study shows optimization of several steps in the protocol for extraction and handling of RNA in renal cortical tissue. RNA extraction and cDNA synthesis can be optimized by the use of the Trizol method and AMV RT, respectively. RNAlater is beneficial for preserving RNA integrity in whole renal cortex during storage and processing, but is not suitable for implementation in routine diagnostic histologic stainings combined with RNA expression studies in dissected biopsy material.
Roos-van Groningen
Marian C
MC
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Eikmans
Michael
M
Baelde
Hans J
HJ
de Heer
Emile
E
Bruijn
Jan A
JA
D000368:Aged; D001706:Biopsy / Q000379:methods*; D015925:Cryopreservation; D018076:DNA, Complementary; D006146:Guanidines; D006801:Humans; D007674:Kidney Diseases / Q000473:pathology* / Q000503:physiopathology; D007678:Kidney Glomerulus / Q000473:pathology* / Q000502:physiology; D008297:Male; D042282:Microdissection; D010636:Phenols; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D012996:Solutions
D018076:DNA, Complementary; D006146:Guanidines; D010636:Phenols; D012333:RNA, Messenger; D012996:Solutions; C411644:trizol