Measuring influenza RNA quantity after prolonged storage or multiple freeze/thaw cycles

In this study, we aim to determine what effects prolonged storage and repeated freeze/thaw cycles have on the stability of influenza A(H1N1)pdm09 (influenza A/H1N1)RNA. Cloned influenza A/H1N1 RNA transcripts were serially diluted from 8.0-1.0 log₁₀ copies/μl. RT-qPCR was used to measure RNA loss in transcripts stored at -80°C, -20°C, 4°C and 25°C for up to 84days or transcripts undergoing a total of 10 freeze/thaw cycles. Viral load was measured in clinical specimens stored at-80°C for three years (n=89 influenza A RNA extracts; n=35 primary specimens) and in 10 clinical specimens from the 2015/2016 influenza season that underwent 7 freeze/thaw cycles. RNA stored at -80°C, -20°C, 4°C and 25°C is stable for up to 56, 56, 21, and 7days respectively or up to 9 freeze/thaw cycles when stored at -80°C. There is no difference in viral load in clinical specimens that have been stored for up to three years at -80°C if they are re-extracted. Similarly, clinical specimens undergoing up to 7 freeze/thaw cycles are stable if they are re-extracted between cycles. Influenza specimens can be stored for up to three years at -80°C or undergo up to 7 freeze/thaw cycles without loss of RNA quantity if re-extracted.