Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds.
Seed polyphenols, polysaccharides and lipids often interfere with or degrade RNA, restricting its yield and quality. Existing RNA isolation methods specifically alleviate one or two of these challenges, but are usually designed for a single species rather than for a broad biodiversity. Many protocols also do not eliminate chromosomal DNA, which causes false positives in gene expression studies. The method reported here addresses all of the above challenges. The concentration of the phenol blocker polyvinylpyrrolidone in the extraction buffer was optimised for use on a broader range of species. DNase I was added to eliminate chromosomal DNA and the timing of this step was optimised. Lipids were removed by centrifugation. Polysaccharides and proteins, including excess DNase I, were separated from RNA during ethanol precipitation of nucleic acids. In seeds of five plant species from four taxonomically distant families, significant amounts of RNA were isolated in less than half the time typically required by previously reported methods. Colorimetric tests showed that the isolated RNA was free from interfering contaminants. In addition, reverse transcription-PCR confirmed that the isolated RNA was of appropriate quality and integrity for gene expression studies.
Birtić
Simona
S
Seed Conservation Department. Royal Botanic Gardens, Kew, Wakehurst Place, RH17 6TN, UK.
Kranner
Ilse
I
D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics; D018532:Pisum sativum / Q000737:chemistry / Q000235:genetics; D010944:Plants / Q000737:chemistry* / Q000235:genetics; D029963:Quercus / Q000737:chemistry / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D018749:RNA, Plant