Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture -: art. no. e64
Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture -: art. no. e64
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DOI：
10.1093/nar/gnh056
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发表时间：
2004-04-01
影响因子：
14.9
通讯作者：
Kauppinen, S
中科院分区：
文献类型：
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作者：
Jacobsen, N;Nielsen, PS;Kauppinen, S
LNA寡核苷酸构成一类双环RNA类似物，其对其互补DNA和RNA靶分子具有异常高的亲和力。我们在这里报告了一种新的方法，用于高效分离完整的poly（A）（+）RNA使用LNA取代的寡（dT）亲和配体，基于LNA-T互补poly（A）tracts的亲和力增加。以LNA-2.T寡核苷酸为亲和探针，直接从4 M硫氰酸胍裂解秀丽隐杆线虫（CaE habditiselegans）虫体提取物及人白血病细胞K562和长春新碱耐药细胞K562/VCR中提取Poly（A）（+）RNA。根据LNA_2.T-A双链体在4 M GuSCN中稳定性的显著增加，我们使用LNA取代的oligo（T）亲和探针获得了比DNA-oligo（dT）选择的mRNA样品高30- 50倍的mRNA产量。此外，LNA_2.T亲和探针在poly（A）结合缓冲液中在50-100 mM NaCl的低盐浓度范围内高度有效地分离poly（A）（+）RNA，如通过从从不同酿酒酵母菌株提取的总RNA样品中选择mRNA库，随后进行北方印迹分析所验证的。最后，我们通过采用预先验证的Taqman测定法分析两种K562细胞系中人mdr 1多药耐药基因的相对表达水平，证明了LNA-寡核苷酸（T）-选择的mRNA在定量实时PCR中的实用性。成功地使用NH 2修饰的LNA 2. T探针分离人mRNA意味着LNA-寡（T）方法可以自动化，用于通过实时PCR进行流线型、高通量表达谱分析，所述实时PCR通过将LNA亲和探针共价偶联到固体、预活化的表面，例如微量滴定板威尔斯孔或磁性颗粒。
LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)(+) RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)(+) RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)(+) RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.