title: A High-Throughput RNA Extraction for Sprouted Single-Seed Barley (Hordeum vulgare L.) Rich in Polysaccharides.
abstract: Germinated seed from cereal crops including barley (Hordeum vulgareL.) 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.
authors.affiliation: 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. Abdur.Rashid@ars.usda.gov.
authors.forename: Abdur
authors.initials: A
authors.lastname: Rashid
authors.affiliation: 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. tom.baldwin@ars.usda.gov.
authors.forename: Thomas
authors.initials: T
authors.lastname: Baldwin
authors.affiliation: Department of Plant and Wild Life Sciences, Brigham Young University (BYU), Provo, UT 84602, USA. michael.c.gines@byu.net.
authors.forename: Michael
authors.initials: M
authors.lastname: Gines
authors.affiliation: 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. Phil.Bregitzer@ars.usda.gov.
authors.forename: Phil
authors.initials: P
authors.lastname: Bregitzer
authors.affiliation: 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.Klos@ars.usda.gov.
authors.forename: Kathy
authors.initials: K
authors.lastname: Esvelt Klos
keywords: barley; cereals; germination; high-quality RNA; qRT-PCR; single-seed extraction; sprouted seed