High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

	High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith
	Yang, Fan 1,2; Tan, Haidong 1; Zhou, Yongjin 1,2; Lin, Xinping 1,2; Zhang, Sufang 1
刊名	molecular biotechnology
	2011-02-01
卷号	47 期号:2 页码:144-151
关键词	RNA extraction Rhodosporidium toruloides Full-length cDNA library
ISSN号	待补充
通讯作者	张素芳
产权排序	1,1
中文摘要	high-quality rna preparation from rhodosporidium toruloides and cdna library construction therewith
英文摘要	oleaginous yeast rhodosporidium toruloides is an excellent microbial lipid producer. therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. high-quality rna extraction from r. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. to obtain an optimal protocol for rna extraction from r. toruloides, four methods were evaluated. large difference in rna yield and quality among these protocols was found. the optimum method was modified rnaiso procedure, where rna was isolated using liquid nitrogen-rnaiso method with salt precipitation and the addition of beta-mercaptoethanol. this method consistently recovered rna in good quality with high yield. around 297 mu g total rna per gram of cells was obtained with an average purity measured as a(260)/a(280) of 2.09. a titer of 10(5) cfu/ml could be harvested to construct a full-length cdna library with the rna sample in this quality. electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. randomly picked clones showed the recombination efficiency at 80%. these results showed that rna of r. toruloides was successfully extracted for the first time using the modified rnaiso method, and the cdna library was appropriate for screening the genes related to lipid accumulation.
学科主题	物理化学
WOS标题词	science & technology ; life sciences & biomedicine
类目[WOS]	biochemistry & molecular biology ; biotechnology & applied microbiology
研究领域[WOS]	biochemistry & molecular biology ; biotechnology & applied microbiology
关键词[WOS]	lipid-accumulation ; oleaginous microorganisms ; plant-tissues ; extraction ; rich ; polysaccharides ; protocol ; culture ; biology
收录类别	SCI
语种	英语
WOS记录号	WOS:000286378600006
公开日期	2012-07-09
内容类型	期刊论文
源URL	[http://159.226.238.44/handle/321008/115574]
专题	大连化学物理研究所_中国科学院大连化学物理研究所
作者单位	1.CAS, Dalian Inst Chem Phys, Div Biotechnol, Dalian 116023, Peoples R China 2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Yang, Fan,Tan, Haidong,Zhou, Yongjin,et al. High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith[J]. molecular biotechnology,2011,47(2):144-151.
APA	Yang, Fan,Tan, Haidong,Zhou, Yongjin,Lin, Xinping,&Zhang, Sufang.(2011).High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith.molecular biotechnology,47(2),144-151.
MLA	Yang, Fan,et al."High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith".molecular biotechnology 47.2(2011):144-151.