|Gines, Michael - Brigham Young University|
|Esvelt Klos, Kathy|
Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2016
Publication Date: 12/22/2016
Citation: Rashid, A., Baldwin, T.T., Gines, M., Bregitzer, P.P., Esvelt Klos, K.L. 2016. A high-throughput RNA extraction for sprouted single-seed malting barley (Hordeum vulgare L.) rich in polysaccharides. Plants. 6(1):1. doi: 10.3390/plants6010001.
Interpretive Summary: Gene expression levels can be measured as the amount of gene-specific RNA. In order to better understand the genes involved in the barley malting process, we have developed a method of high-throughput RNA extraction for 4-day old sprouted seed. This tissue is technically challenging because the sprouted seed is saturated with starch that interferes with most RNA extraction protocols. Our protocol is suitable for high-throughput laboratory applications because it is fast, simple and produces ample high-quality RNA for investigations of gene expression. Understanding which genes are expressed during the malting process may aid in developing improved barley varieties for the malting and brewing industries.
Technical Abstract: 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 increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat.