DICP OpenIR
Subject Area物理化学
High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith
Yang, Fan1,2; Tan, Haidong1; Zhou, Yongjin1,2; Lin, Xinping1,2; Zhang, Sufang1; Zhang SF(张素芳)
KeywordRna Extraction Rhodosporidium Toruloides Full-length Cdna Library
Source PublicationMOLECULAR BIOTECHNOLOGY
2011-02-01
ISSN待补充
DOI10.1007/s12033-010-9322-1
Volume47Issue:2Pages:144-151
Indexed BySCI
SubtypeArticle
Department18
Funding Project1816
Contribution Rank1,1
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS KeywordLIPID-ACCUMULATION ; OLEAGINOUS MICROORGANISMS ; PLANT-TISSUES ; EXTRACTION ; RICH ; POLYSACCHARIDES ; PROTOCOL ; CULTURE ; BIOLOGY
AbstractHigh-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith; 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.
Language英语
WOS IDWOS:000286378600006
Citation statistics
Cited Times:4[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/115574
Collection中国科学院大连化学物理研究所
Corresponding AuthorZhang SF(张素芳)
Affiliation1.CAS, Dalian Inst Chem Phys, Div Biotechnol, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China
Recommended Citation
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,&张素芳.(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.
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