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Letters to the Editor |
Center for Biological Safety, Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany, Fax 49-30-45472605, E-mail: kurtha{at}rki.de
To the Editor:
When freezing facilities for biological materials are not available, there is a need for fixatives that allow the examination of single samples for morphological as well as antigen, antibody, or nucleic acid analyses. A widely used stabilization buffer, RNAlaterTM RNA Stabilization Reagent (Qiagen), has been assessed in clinical tissues and compared with snap-freezing (1) for use in RNA preservation for quantitative RNA expression studies and histologic and immunohistochemical investigations(2). Protection of flavivirus antigen was demonstrated by antigen-capture ELISA(3), and antibody assays have been completed using RNAlater-preserved materials(4). According to the manufacturer the lack of RNAlater toxicity makes the product user-friendly for sample transportation, but biological safety may still be a concern with some RNAlater-treated specimens. Picornavirus, rhabdovirus, and HIV in cell culture supernatants retain infectivity after RNAlater stabilization(5). The study described here evaluates the risk of remaining poxvirus infectivity from cultured cells and tissue specimens fixed with RNAlater at various temperatures mimicking field conditions.
Semiconfluent Hep2 cells were infected with an orthopoxvirus (vaccinia virus, VV, strain NYCDH) at a multiplicity of infection of 0.1 and incubated for 24 h. After removal of the supernatant, cells were covered with RNAlater and stored at 20 °C in a biological safety cabinet. After 5 days, vaccinia virus–infected cells were harvested, freeze-thawed to release intracellular virus, and centrifuged at 15 000g; then the pellet was resuspended in growth medium (DMEM, Gibco). Concurrently, to assess retention of vaccinia virus infectivity in RNAlater–preserved organ tissue, embryonated chicken eggs were infected with 100 plaque forming units (PFU) of vaccinia virus and dissected after 3 days of incubation. The chick embryo livers were removed and placed into RNAlater. To duplicate field conditions the submerged tissue was incubated at 4, 20, and 37 °C. After 5 days, RNAlater-treated tissues were washed once in PBS and homogenized in 1 mL growth medium. Aliquots were processed for standard plaque assay and DNA isolation (Qiagen QIAamp DNA Blood Mini Kit). DNA was amplified by PCR with previously described primer sequences and cycling conditions using the orthopoxvirus (OPV)-specific rpo18-assay (6) in a real-time PCR sequence detection system (Stratagene Mx3005PTM). For virus quantification the ratio of infectious vaccinia virus particles (PFU) and viral genomic DNA [genome equivalents (GE)] were calculated referring to 104 GE of a chicken-specific reference gene [TATA-box binding protein (TBP) NM_205103]. Primers and hybridization probe for TBP were as follows: gTgTCCACggTgAATCTTgg (galTBP F, 57.3 °C), TgCATTCTAACATACTTCTCTTACCTTg (galTBP R, 56.5 °C), F-CgTgCCCgAAATgCTgAATATAATCCCA q (galTBP TM, 67.9 °C, F: 6-FAM, q: dark quencher). Results from RNAlater-treated samples were compared with data from liver specimens frozen at –20 °C without RNAlater treatment. After 5 days at 20 °C in RNAlater, the treated cell culture samples retained 5.9 x 109 PFU/L of vaccinia virus, compared with 1.7 x 1010 PFU/L for the untreated controls. Similarly, vaccinia virus could be detected in all infected chicken embryo liver samples (Fig. 1
). Infectivity was inversely proportional to storage temperatures (37 °C, 
4 log10 decrease in titer; 20 °C, 2 log10 decrease; 4 °C, 1 log10 decrease). Vaccinia virus DNA remained stable at all incubation temperatures during 5 days of storage compared with the –20 °C frozen control specimen.
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To date, more than 43 publications since 2000 describe the advantages of RNAlater as a protective buffer suitable for nucleic acid amplification techniques in various scientific fields. RNAlater is proposed for RNA protection in tissue, mammalian cells, human saliva, and blood (Stabilization solutions 07/2006; http://www.qiagen.com). RNAlater stabilization is widely replacing snap-freezing for the conservation of DNA and RNA in tissue specimens collected in field trials. Blacksell et al. (3) encouraged its use for stabilizing RNA viruses for shipment and diagnostic investigations. However, a potential health risk from RNAlater-treated blood and saliva samples was identified in virus cell cultures in which viral infectivity was preserved in supernatants(5). The study reported here reveals that both cultured cells and tissue specimen had vaccinia virus infectivity persistent for 
5 days at all tested temperatures when stored in RNAlater. Consequently, RNAlater-treated clinical tissue can present a biohazard to laboratory workers. Because RNAlater is readily used for field sample transportation, laboratory assistants could falsely interpret RNAlater as a disinfectant fixative. Therefore, RNAlater-treated specimens should be marked as potentially infective materials. This study also demonstrates an important useful feature through which both nucleic acid integrity and viral cell culture infectivity can be retained in a single specimen transported at ambient temperatures without maintaining a cooling chain, even under tropical conditions.
In summary, because of remaining infectivity within RNAlater-treated clinical or field samples, all personnel handling these specimens should be aware of the potential disease risk.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: None declared.
Acknowledgments: I am grateful to Delia Barz and Jung-Won Sim-Brandenburg for excellent technical assistance and Dr. Andreas Nitsche for designing TBP primers and hybridization probe.
References
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