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70% ethanol preserves mycobacterial RNA from culture more efficiently than GTC-TCEP

L Kraußer(1,2,3) M Van Dyck-Lippens(1) R Balde(1) R Reenaers(1) L Rigouts(1,2) B C de Jong(1) S M Braet(1)

1:Institute of Tropical Medicine (ITM), Antwerp, Belgium; 2:University of Antwerp, Antwerp, Belgium; 3:Research Foundation Flanders (FWO), Brussels, Belgium

The efficient preservation of mycobacterial RNA molecules is crucial both for research and clinical applications. For Mycobacterium tuberculosis RNA-based viability assessment, e.g. RS ratio, has a substantially faster turnaround time than conventional phenotypic drug susceptibility testing. Searching for an alternative to the widely used RNA fixating buffer GTC-TCEP which requires storage at ultra-low temperatures, we compared it to 70 % ethanol fixation. For this, a suspension of cultured Mtb H37Ra was mixed with either 70 % ethanol or 4.5 M GTC-TCEP and stored in triplicate for up to 12 months at -80 °C, -20 °C, 4 °C or 30 °C. RNA extracts were analysed in terms of purity and integrity and reverse transcribed before the targets iclesxA16S and sigA were quantified by Mtb specific qPCR. Results show that both quantity and integrity of RNA were consistently higher when cultured Mtb was stored in 70 % ethanol. The normalised RNA quantity determined by qPCR remained comparable to fresh samples for up to 12 months at 4 °C when stored in 70 % ethanol. Storage at 30 °C resulted in heavily degraded RNA regardless of the fixative. Based on these findings we propose 70 % ethanol and 4 °C as adequate storage conditions for RNA preservation in cultured Mtb for up to six months. This could be a possible low-cost alternative for the preservation of samples collected in field conditions. However, a confirmation of these findings for clinical samples is still necessary to translate the findings to a clinical setting. 

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