- 期刊
Isolation of Functional RNA from Different Tissues of Tomato Suitable for Developmental Profiling by Microarray Analysis
從番茄不同組織分離功能性RNA並以微矩陣方法進行發育圖譜分析
摘要
我們詳述一個從番茄不同發育時期之營養及生殖組織中分離高品質RNA的方法，所獲得之RNA可應用在微矩陣分析上。這個方法基本上是依照TRIzol方法並加入氯化鋰(LiCl)沉澱及DNase處理。使用這個方法，可以獲得產量較多之高品質及完整的RNA，其品質以分光光度儀、膠體電泳及螢光儀驗證之。我們進一步以下述方法闡明所獲得的完整RNA具有功能：(1)這些RNA以螢光試劑標記後用作探針，與番茄cDNA微矩陣雜交，找到了果實成熟時期不同表現之基因；(2)利用反轉錄聚合酶鏈反應(RT-PCR)並以這些RNA作為模板，我們成功地選殖了番茄類胡蘿蔔素生合成路徑的數個關鍵基因，包括phytoene synthase (PSY)、phytoene desaturase (PDS)、ζ-carotene desaturae (ZDS)及lycopeneβ-cyclase (LCY)等；及(3)這些RNA可以供作構築cDNA基因庫。
並列摘要
An efficient and reproducible method is described for isolating high-quality RNA suitable for microarray analysis from vegetative and reproductive tissues of tomato plants at different stages of growth. This method was based on TRIzol method followed by lithium chloride (LiCl) precipitation and DNase treatment. Using this method, high yields of high-quality and undegraded RNA were obtained as confirmed by spectrophotometric method, gel electrophoresis and fluorescent quality control. The integrity and functionality of RNA isolated by this procedure have been further demonstrated as probe in tomato cDNA microarrays for identification of differentially expressed genes during fruit ripening, as template for cloning full-length cDNAs encoding phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS) and lycopene β-cyclase (LCY) in the carotenoid biosynthesis pathway by the reverse transcription-polymerase chain reaction (RT-PCR), and as material for cDNA library construction.
延伸閱讀
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