An improved method of isolation of high quality total RNA from purple-fleshed sweet potato, Ipomoea batatas (L.) Lam.
To isolate high quality total RNA from plants of purple-fleshed sweet potato (Ipomoea batatas L. Lam) is difficult because these plants contain high levels of carbohydrates and pigment compounds. In this study, we developed a quick and reliable method for total RNA extraction. Using this extraction protocol, which was derived from the traditional CTAB method with modification, we obtained high quality total RNA from all the tested tissues including leaf blade, petiole, stem, fibrous root, thick root, and storage root. Spectrophotometric analysis, together with agarose gel electrophoresis analysis of extracted total RNA, indicated that the total RNA was perfectly intact and the yields were all more than 0.2 mg/g fresh weight of different tissues. A metallothionein-like protein gene (G14) fragment was successfully amplified by reverse-transcription-PCR and cDNAs of two target genes (F3'H and DFR) were isolated by the method of 3'-RACE and 5'-RACE using the first-strand 3'-RACE-ready and 5'-RACE-ready cDNA as templates which were synthesized from the total RNA extraction, indicating that the total RNA extraction was suitable for further molecular analysis.
Zhou
Wei
W
Guangdong Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, P. R. China.
Gong
Yifu
Y
Feng
Qili
Q
Gao
Feng
F
D005591:Chemical Fractionation / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D027723:Ipomoea batatas / Q000737:chemistry / Q000235:genetics*; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics; D018517:Plant Roots / Q000737:chemistry / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018749:RNA, Plant