Rehydration conditions for isolation of high quality RNA from the lichen Lobaria pulmonaria

Abstract

BackgroundThe poikilohydric nature of lichens enables them to survive repeated episodes of desiccation by utilizing water when it becomes available. During rehydration, RNA-degrading endonucleases may be released, reducing RNA quantity and quality. Re-generation of a steady-state condition where RNA quantity and quality no longer fluctuate establishes a framework for development of new hypotheses for future investigations. Using Lobaria pulmonaria as a model species, the objective of this study was to compare the effect of different rehydration conditions on the quantity and quality of RNA from the rehydrated thallus.FindingsSpectrophotometric measurements of total RNA and cDNA were performed for samples prepared from dry lichen or lichen after rehydration (0.5 h, 1 h, 2 h, 4 h or 24 h), with limited light and dark conditions, and at three temperatures (15°C, 20°C or 32°C) for some of these conditions. The results showed that rehydration of the thallus for 4 h at 20°C in light yielded the highest concentration and quality of RNA. A higher RNA concentration was obtained in light than in dark conditions, but the RNA quality was unaffected.ConclusionsThis study suggests that allowance of 4 h for thallus rehydration should be adequate to ensure complete recovery of transcription. After 4 h at 20°C further studies can be carried out on the RNA in this model species.

Stressed out: the effects of heat stress and parasitism on gene expression of the lichen-forming fungus Lobaria pulmonaria

“…The samples taken after exposure to 4 °C and 15 °C were immediately used for RNA extraction, while the 25 °C samples were stored overnight at −80 °C. Successful RNA extractions for Lobaria pulmonaria have been reported using TRI Reagent (Doering et al 2014), therefore this extraction chemistry was used. Samples were homogenized in TRI Reagent (Sigma Aldrich) using Tissue Lyser II (Qiagen) with a 3 mm stainless steel polishing bead (Kugel Pompel, Austria).…”