TY - JOUR

T1 - Extraction of Microbial and Host DNA, RNA, and Proteins from Oak Bark Tissue

AU - Broberg, Martin

AU - McDonald, James

PY - 2019/2/5

Y1 - 2019/2/5

N2 - The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota.

AB - The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota.

U2 - 10.3390/mps2010015

DO - 10.3390/mps2010015

M3 - Article

VL - 2

JO - Methods and Protocols

JF - Methods and Protocols

SN - 2409-9279

IS - 1

M1 - 15

ER -