Development of an efficient protocol of RNA isolation from recalcitrant tree tissues.
Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities. It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues.
Wang
Xuchu
X
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Chinese Academy of Sciences, Beijing, 100093, People's Republic of China.
Tian
Weimin
W
Li
Yinxin
Y
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D015152:Blotting, Northern; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D017931:DNA Primers / Q000235:genetics; D004492:Edetic Acid; D017343:Genes, Plant; D032125:Litchi / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D009994:Osmolar Concentration; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification*; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D012521:Sarcosine / Q000031:analogs & derivatives; D014197:Trees / Q000235:genetics*
D002593:Cetrimonium Compounds; D017931:DNA Primers; D018749:RNA, Plant; C025231:sarkosyl; D004492:Edetic Acid; D012260:Ribonucleases; D012521:Sarcosine; D000077286:Cetrimonium