RNA extraction from mycotoxigenic filamentous fungi - Bertin Instruments

    Microbiology

    RNA extraction from mycotoxigenic filamentous fungi

    Sources: Applied Mycology Group, Cranfield Health, Cranfield University, Cranfield, Bedfordshire, UK

    Context

    Filamentous fungi cell wall and endogenous RNase activity often hindered the study of the molecular mechanisms behind secondary metabolites. Toxigenic secondary metabolites affect human health and represent a risk in the food chain system. The conditions in which mycotoxins are produced are still not completely understood especially the molecular mechanism that trigger their production. The extraction of high quality RNA in good amounts was therefore critical to further evaluate the conditions in which mycotoxigenic gene clusters are activated. Aspergillus flavus NRRL 3357 was used to evaluate the RNA extraction protocol. A manual grinding method using mortar and pestle was compared to different bead beating homogenizers using different beads [1].

     

    [1] G.M. Leite, N. Magan, A. Medina. 2012.Comparison of different bead-beating RNAextraction strategies: an optimized method for filamentous fungi. Journal ofMicrobiological Methods, 88, 413-418.

    Results

    Significant differences were found between the total RNA amounts isolated using the Precellys®24 homogenizer and the manual system (p-value=0.0072). Furthermore the use of glass beads resulted in a yield around 3 times higher than using the traditional method of the mortar and pestle (Figure 1). The high quality of the RNA as also achieved as an example the electropherogram of a high quality RNA sample extracted using the 0.5 mm glass beads (Figure 2).

     

    Total RNA yield average per 100 mg of initial biomass and standard deviation comparing different beads with the manual method. Key to treatments: CK – Zirconnium Oxide, VK – Glass; followed by the bead size code 01 – 0.1 mm, 05 – 0.5 mm, 14 – 1.4 mm, 28 – 2.8 mm.

    Total RNA yield average per 100 mg of initial biomass and standard deviation comparing different beads with the manual method. Key to treatments: CK – Zirconnium Oxide, VK – Glass; followed by the bead size code 01 – 0.1 mm, 05 – 0.5 mm, 14 – 1.4 mm, 28 – 2.8 mm.

     

    Electropherogram of a good quality RNA sample extracted using Precellys at room temperature. The RQI of this sample is 9.7.

    Electropherogram of a good quality RNA sample extracted using Precellys at room temperature. The RQI of this sample is 9.7.

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