RNA isolation from biological soil crusts: methodological aspects

Biological soil crust (BSC) communities can be found in almost all environments except for the tropics. These microbial mats are especially predominant in ecosystems, which exhibit harsh conditions, in which they play a key role as primary producers. Studying their metatranscriptomic data enables scientists to shed light on taxa composition, the interactions between those organisms and their ability to cope with abiotic stressors in the extreme environments, e.g.the polar regions. The basis of a successful metatranscriptomic analysis is the isolation of pure RNA of high quality and integrity. Nucleic acid extraction from soil samples is challenging due the diverse chemical and physical properties of soil. Humic substances are often co-extracted and, subsequently, contaminate the sample. In this study, different RNA extraction techniques were tested and evaluated for the isolation of high quality RNA from four BSC samples, three isolated in Germany and one polar BSC: a CTAB based protocol, the Spectrum™ Plant Total RNA Kit and the Precellys Plant RNA Kit. The CTAB based method provides high quality RNA at a low yield and with an average degree of contamination for all tested samples. RNA obtained by using the Spectrum™ Plant Total RNA Kit is of high quality with only little contamination but only half of the samples could be processed successfully. A high throughput approach is the Precellys Plant RNA Kit resulting in a fair RNA quality but with the highest level of contamination. Furthermore, only three out of four samples yielded any results. Further purification is often necessary as DNA and humic substances often cannot be easily removed. Using enzymatic digestion DNA can be specifically cleaved while humic substances can be separated from the RNA fraction by using either gel filtration or RNA affinity binding columns.