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RNAi質(zhì)粒載體構(gòu)建|實(shí)驗(yàn)技術(shù)服務(wù)

時(shí)間:2015/6/2閱讀:676
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關(guān)鍵詞:RNAi質(zhì)粒載體構(gòu)建|實(shí)驗(yàn)技術(shù)服務(wù)
簡(jiǎn)介:世界*品牌RNAi質(zhì)粒載體構(gòu)建|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
RNAi質(zhì)粒載體構(gòu)建|實(shí)驗(yàn)技術(shù)服務(wù)——pCzn1質(zhì)粒+Arctic Express宿主菌低溫表達(dá)體系,。
我們具備豐富的蛋白表達(dá)設(shè)計(jì)經(jīng)驗(yàn)及實(shí)驗(yàn)操作技巧,,承諾客戶不成功不收費(fèi),。在獲得重組蛋白產(chǎn)品的同時(shí),,我們也會(huì)提供相應(yīng)的原始數(shù)據(jù)和原始圖片,不需要再額外花費(fèi)精力重復(fù)實(shí)驗(yàn),。另一方面,我們所有的實(shí)驗(yàn)數(shù)據(jù)真實(shí)可信,,我們提供原始的表達(dá)菌株和克隆質(zhì)粒,,客戶可以依據(jù)我們的實(shí)驗(yàn)報(bào)告重復(fù)我們的實(shí)驗(yàn)結(jié)果。
產(chǎn)品品牌:   http://www.,。,。com/goodsid/fenleiyi/2868603/1.html    
測(cè)序?qū)嶒?yàn)流程:
近年來的研究表明,一些短片斷的雙鏈RNA可以通過促使特定基因的mRNA降解來、特異的阻斷體內(nèi)特定基因表達(dá),誘使細(xì)胞表現(xiàn)出特定基因缺失的表型, 稱為RNA干擾(RNA interference,RNAi).siRNA(small interfering RNAs)就是這種短片斷雙鏈RNA分子,能夠以序列同源互補(bǔ)的mRNA為靶目標(biāo),降解特定的mRNA.RNAi的發(fā)現(xiàn)具有劃時(shí)代的意義,它不僅深入揭示 了細(xì)胞內(nèi)基因沉默的機(jī)制,而且它還是后基因組時(shí)代基因功能分析的有力工具,極大地促進(jìn)了人類揭示生命奧秘的進(jìn)程.現(xiàn)在越來越多的研究人員開始采用RNAi 來研究生物體的基因表達(dá).RNAi技術(shù)可廣泛應(yīng)用到包括功能基因組學(xué),藥物靶點(diǎn)篩選,細(xì)胞信號(hào)傳導(dǎo)通路分析,疾病治療等等.
目前為止較為常用的5種制備siRNAs的方法包括:
·化學(xué)合成
·體外轉(zhuǎn)錄
·長(zhǎng)片斷dsRNAs經(jīng)RNase III 類降解 (e.g. Dicer, E. coli, RNase III)
·siRNA表達(dá)載體或者病毒載體在細(xì)胞中表達(dá)siRNAs
·PCR制備的siRNA表達(dá)框在細(xì)胞中表達(dá)
獲得高純度的siRNA產(chǎn)物是進(jìn)行實(shí)驗(yàn)的*步,而轉(zhuǎn)染的效率則是非常關(guān)鍵的因素.
RNAi表達(dá)載體的構(gòu)建
1. 目的基因的確定
2,、設(shè)計(jì)siRNA靶序列
在制備siRNA 前都需要單獨(dú)設(shè)計(jì)siRNA序列.研究發(fā)現(xiàn)對(duì)哺乳動(dòng)物細(xì)胞,zui有效的siRNAs是21-23個(gè)堿基大小,、3’端有兩個(gè)突出堿基的雙鏈RNA;而對(duì)非哺乳動(dòng)物,比較有效的是長(zhǎng)片段dsRNA.siRNA的序列專一性要求非常嚴(yán)謹(jǐn),與靶mRNA之間一個(gè)堿基錯(cuò)配都會(huì)顯著削弱基因沉默的效果.
(1),、選擇siRNA靶位點(diǎn):
從轉(zhuǎn)錄AUG起始密碼子開始,搜尋下游AA序列,記錄跟每個(gè)AA 3’端相鄰的19個(gè)核苷酸作為候選的siRNA靶位點(diǎn).有研究結(jié)果顯示GC含量在30%—50%左右的siRNA要比那些GC含量偏高的更為有效. Tuschl等建議在設(shè)計(jì)siRNA時(shí)不要針對(duì)5'和3'端的非編碼區(qū)(untranslated regions,UTRs),原因是這些地方有豐富的調(diào)控蛋白結(jié)合區(qū)域,而這些UTR結(jié)合蛋白或者翻譯起始復(fù)合物可能會(huì)影響siRNP核酸內(nèi)切酶復(fù)合物結(jié) 合mRNA從而影響siRNA的效果.
(2),、序列同源性分析:
將潛在的序列和相應(yīng)的基因組數(shù)據(jù)庫(kù)(人,或者小鼠,大鼠等等)進(jìn)行比較,排除那些和其他編碼序列/EST同源的序列.例如使用BLAST( www.ncbi.nlm.nih.gov/BLAST/)選出合適的目標(biāo)序列進(jìn)行合成.并非所有符合條件的siRNA都一樣有效,其原因還不清楚,可能是位置效應(yīng)的結(jié)果,因此對(duì)于一個(gè)目的基因,一般要選擇3-5個(gè)靶位點(diǎn)來設(shè)計(jì)siRNA.

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