A High-Throughput RNA Extraction for Sprouted Single-Seed Barley (Hordeum vulgare L.) Rich in Polysaccharides
Abdur Rashid,
Thomas Baldwin,
Michael Gines,
Phil Bregitzer,
Kathy Esvelt Klos
Affiliations
Abdur Rashid
United States Department of Agriculture—Agriculture Research Service (USDA-ARS), Small Grains and Potato Germplasm Research Unit, 1691 S. 2700 W., Aberdeen, ID 83210, USA
Thomas Baldwin
United States Department of Agriculture—Agriculture Research Service (USDA-ARS), Small Grains and Potato Germplasm Research Unit, 1691 S. 2700 W., Aberdeen, ID 83210, USA
Michael Gines
Department of Plant and Wild Life Sciences, Brigham Young University (BYU), Provo, UT 84602, USA
Phil Bregitzer
United States Department of Agriculture—Agriculture Research Service (USDA-ARS), Small Grains and Potato Germplasm Research Unit, 1691 S. 2700 W., Aberdeen, ID 83210, USA
Kathy Esvelt Klos
United States Department of Agriculture—Agriculture Research Service (USDA-ARS), Small Grains and Potato Germplasm Research Unit, 1691 S. 2700 W., Aberdeen, ID 83210, USA
Germinated seed from cereal crops including barley (Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the excess polysaccharides. Here, we compare the current methods for RNA extraction applicable to germinated barley tissue. We found that although some of these standard methods produced high-quality RNA, the process of extraction was drastically slow, mostly because the frozen seed tissue powder from liquid N2 grinding became recalcitrant to buffer mixing. Our suggested modifications to the protocols removed the need for liquid N2 grinding and significantly increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat.
