Preservation of RNA and destruction of infectivity in microdissected brain tissues of Lewis rats infected with the Borna disease virus.
Laser microdissection combined with real-time RT-PCR presents an advanced tool to quantify particular RNA species in defined tissue areas. Dealing with infectious tissue samples increases the need to overcome the risk of infectivity and contamination during laser microdissection. Here, an useful method to control infectivity of frozen brain sections infected with the Borna disease virus (BDV), an enveloped RNA virus, is described. Various pre-treatments were applied prior to laser microdissection and subsequent real-time RT-PCR. Brain sections were incubated with Vennotrade mark Vet 1 super 1% or 70% ethanol for 30, 60 and 90min, followed by quantification of infectious virus and RNA recovery using laser microdissection. Total RNA specific for the BDV nucleoprotein (BDV-N) and the cellular genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate-ubiquinone reductase (SDHA) and hypoxanthine phosphoribosyl-transferase-1 (HPRT) was measured by real-time RT-PCR and compared to BDV-infected control samples. After 30 min incubation with both disinfectants, no infectious virus was isolated, while sufficient cDNA copy numbers were amplified. As tissue morphology was best preserved after ethanol treatment, 30min incubation with 70% ethanol was selected as the method of choice to prevent infectivity of BDV. This procedure represents a suitable pre-treatment option to ensure adequate safety of virus infected central nervous system tissue.
Porombka
Doris
D
Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany. doris.porombka@tiho-hannover.de
Herzog
Sibylle
S
Baumgärtner
Wolfgang
W
Herden
Christiane
C
D000818:Animals; D001891:Borna disease virus / Q000235:genetics* / Q000472:pathogenicity; D001921:Brain / Q000473:pathology / Q000821:virology*; D018076:DNA, Complementary / Q000235:genetics; D042963:Electron Transport Complex II / Q000235:genetics; D018628:Gene Dosage; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D042282:Microdissection; D009698:Nucleoproteins / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis*; D051381:Rats; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D009698:Nucleoproteins; D012367:RNA, Viral; D042963:Electron Transport Complex II; D007041:Hypoxanthine Phosphoribosyltransferase