Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens
JOURNAL OF INFECTIOUS DISEASES
Authors: Erickson, Bobbie R.; Sealy, Tara K.; Flietstra, Tim; Morgan, Laura; Kargbo, Brima; Matt-Lebby, Victor E.; Gibbons, Aridth; Chakrabarti, Ayan K.; Graziano, James; Presser, Lance; Flint, Mike; Bird, Brian H.; Brown, Shelley; Klena, John D.; Blau, Dianna M.; Brault, Aaron C.; Belser, Jessica A.; Salzer, Johanna S.; Schuh, Amy J.; Lo, Michael; Zivcec, Marko; Priestley, Rachael A.; Pyle, Meredith; Goodman, Christin; Bearden, Scott; Amman, Brian R.; Basile, Alison; Bergeron, Eric; Bowen, Michael D.; Dodd, Kimberly A.; Freeman, Molly M.; McMullan, Laura K.; Paddock, Christopher D.; Russell, Brandy J.; Sanchez, Angela J.; Towner, Jonathan S.; Wang, David; Zemtsova, Galina E.; Stoddard, Robyn A.; Turnsek, Maryann; Guerrero, Lisa Wiggleton; Emery, Shannon L.; Stovall, Janae; Kainulainen, Markus H.; Perniciaro, Jamie L.; Mijatovic-Rustempasic, Slavica; Shakirova, Gulchekhra; Winter, Jorn; Sexton, Christopher; Liu, Feng; Slater, Kimetha; Anderson, Raydel; Andersen, Lauren; Chiang, Cheng-Feng; Tzeng, Wen-Pin; Crowe, Samuel J.; Maenner, Matthew J.; Spiropoulou, Christina F.; Nichol, Stuart T.; Stroher, Ute
Abstract
During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.
Optimized Biobanking Procedures for Preservation of RNA in Tissue: Comparison of Snap-Freezing and RNAlater-Fixation Methods
BIOPRESERVATION AND BIOBANKING
Authors: Hentze, Julie L.; Kringelbach, Tina M.; Novotny, Guy W.; Hamid, Bushra H.; Ravn, Vibeke; Christensen, Ib J.; Hogdall, Claus; Hogdall, Estrid
Abstract
Introduction: Personalized treatment, supported by biomarkers, would improve survival of ovarian cancer patients. RNA molecules are potentially important biomarkers. The Danish CancerBiobank provides an infrastructure for handling and storage of biological material, including RNA, from Danish cancer patients. The aim of this study was to investigate the effects of handling-time and fresh-freezing versus RNAlater (R) fixation on RNA degradation in solid tissue from pelvic mass samples. Materials and Methods: We evaluated RNA quality in surgical tissue from patients with a pelvic mass. Corresponding samples were either fresh-frozen or fixed in RNAlater, at eight different time points after the surgery. Integrity was measured using a bioanalyzer, and the amount and quality were further investigated by quantitative reverse transcription-polymerase chain reaction measuring the expression of housekeeping genes B2M and HPRT1. Results: Our results show that tissue RNA is stable up to at least 180 minutes after the surgery, as the quality was comparable to the quality of RNA handled immediately. Likewise, patient RNA was of acceptable quality after both fresh-frezing and RNAlater fixation, but RNAlater fixation was slightly more effective for RNA preservation. Discussion and Conclusion: Our data suggest that RNA in pelvic mass samples is relatively stable. Knowledge about RNA stability is an important prerequisite for research in RNA biomarkers, where the challenge is to balance the need for careful RNA handling and storage with the need for effective large-scale biobanking in a busy clinical setting where patient treatment is the main priority.
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