siPOOLs用于藥物靶點(diǎn)篩選研究（RNAi）

對(duì)于靶向基因沉默，RNA干擾（RNAi)具有易于操作、快速結(jié)果、高效率、廣泛適用于各種細(xì)胞類型的多重優(yōu) 勢(shì)。其具有瞬轉(zhuǎn)效應(yīng)和劑量依賴效應(yīng)，這點(diǎn)與小分子非常相似。然而，目前的siRNA試劑其特異性和基因沉默 效率可變，阻礙了它們作為藥物發(fā)現(xiàn)和基因研究工具的應(yīng)用。

siTOOLs Biotech由Michael Hannus博士和Gunter Meister教授于2013年9月成立，由德國(guó)雷根斯堡大學(xué) 和Intana Bioscience制藥服務(wù)公司聯(lián)合運(yùn)營(yíng)。目前推出有3個(gè)產(chǎn)品系列：（1）siPOOLs：一種siRNA混合物，有 效增加基因沉默特異性，“稀釋”傳統(tǒng)RNA干擾試劑的脫靶效應(yīng)。（2）raPOOLs：一種穩(wěn)健的RNA親和純化方 案，適用于基因功能和相互作用的生物化學(xué)研究。（3）riboPOOLs：適合任意物種的核糖體RNA去除試劑，高效且經(jīng)濟(jì)。

可靠表型的特異性基因沉默工具

siPOOLs是一款經(jīng)過優(yōu)化設(shè)計(jì)的高復(fù)雜性的包含30條siRNA的混合物，經(jīng)證明可有效消除脫靶效應(yīng)，并提高結(jié)果的可靠性（Hannus et al., 2014）。 Pack Hunter (pooling) 方法通過將當(dāng)個(gè)siRNA的 濃度稀釋到刺激表型的閾值以下來對(duì)抗單個(gè)siRNA的脫靶。 借助專有的設(shè)計(jì)算法，siPOOLs中的siRNA序列經(jīng) 過優(yōu)化，以實(shí)現(xiàn)最大的轉(zhuǎn)錄本覆蓋率，高效雜交并 對(duì)旁系同源基因進(jìn)行過濾，從而實(shí)現(xiàn)高效和特異性 的基因沉默。

siPOOLs產(chǎn)品優(yōu)勢(shì)

使用簡(jiǎn)單快捷：siPOOLs與多種轉(zhuǎn)染試劑兼容，幾天內(nèi)就能看 到結(jié)果。

高度特異性且有效：siPOOLs在標(biāo)準(zhǔn)細(xì)胞系中將脫靶率降低5-25 倍，且在1-3 nM下實(shí)現(xiàn)基因敲低率≥70%。

一致的表型：與siRNA相反，由序列非依賴性的siPOOL產(chǎn)生 的表型高度一致。

確保經(jīng)過檢驗(yàn)：通過RT-qPCR進(jìn)行siPOOL驗(yàn)證，如果在 轉(zhuǎn)染下敲低率低于70%，則有可能重新設(shè)計(jì)。

使用注釋進(jìn)行定制設(shè)計(jì)：專業(yè)的設(shè)計(jì)，確保優(yōu)化熱力學(xué)特性且避免旁系 同源基因。

HPLC純化且無毒：所有siPOOL均經(jīng)過HPLC純化，可降低污染物 和副作用的風(fēng)險(xiǎn)。

關(guān)鍵問題：siRNAs的脫靶效應(yīng)

科學(xué)家們一直在使用RNA干擾（RNAi）作為研究基因功能的快速有效的工具。然而，短干擾RNA（siRNA）的脫靶效應(yīng)和可變性能仍然是一個(gè)令人頭疼的缺點(diǎn)，在驗(yàn)證工作中消耗了寶貴的時(shí)間和資源。

siRNA通常與靶RNA轉(zhuǎn)錄本互補(bǔ)結(jié)合，通過RNAi機(jī)制指導(dǎo)其降解。脫靶效應(yīng)主要是由siRNA模擬內(nèi)源性 基因調(diào)節(jié)因子microRNA（miRNA）引起的。由于miRNA只需要6個(gè)堿基種子匹配到3'非翻譯區(qū)（UTR）即可觸 發(fā)轉(zhuǎn)錄本下調(diào)，因此siRNA在通過這種機(jī)制處理時(shí)可以改變?cè)S多意外靶標(biāo)的表達(dá)。

siPOOLs如何提高特異性

單個(gè)siRNA或含有3-4個(gè)siRNA的低復(fù)雜度siRNA庫(kù)，經(jīng)常擊中多個(gè)脫靶基因并表現(xiàn)出易變的靶基因敲低。 siPOOL是高度復(fù)雜且確定的30個(gè)siRNA池，每個(gè)siRNA以皮摩爾工作濃度存在。因此， · 稀釋了每個(gè)siRNA的脫靶特征，提高了靶向特異性。 · 確保了靶基因的協(xié)同敲低，產(chǎn)生更穩(wěn)健、更可靠的結(jié)果。

使用siPOOL具有更高的特異性

特異性好的試劑應(yīng)僅影響其靶標(biāo)基因。

HeLa細(xì)胞中微陣列的表達(dá)譜分析顯示，單個(gè)siRNA可以誘導(dǎo)許多脫靶基因（紅點(diǎn)），而針對(duì)同一靶基因（綠 點(diǎn)）并包含非特異性siRNA的siPOOL大大降低了脫靶效應(yīng)。

通過siPOOL實(shí)現(xiàn)更好的重現(xiàn)性和有效的敲低效率

普通siRNA的敲低效率差異很大。

與單個(gè)siRNA相比，針對(duì)同一基因的siPOOLs具有相似的敲低效率，表明具有更高的穩(wěn)健性和可重復(fù)性（左 圖和中圖：靶RNA水平的實(shí)時(shí)定量PCR測(cè)量的相關(guān)圖）。 siPOOLs也表現(xiàn)出有效的基因敲低。在常用的細(xì)胞系中，許多基因通常在1 nM濃度下實(shí)現(xiàn)75-98%的基 因敲低效率（右圖）。

您可以信賴的表型

如果RNAi效果可靠且特異，靶向同一基因的兩種試劑應(yīng)產(chǎn)生相似的表型。

每個(gè)基因分別使用兩個(gè)siPOOL池，一個(gè)來自我們的人類激酶siPOOL文庫(kù)，一個(gè)是市售的每個(gè)基因含3條 siRNAs的文庫(kù)，科學(xué)家在A549細(xì)胞中篩選了36個(gè)基因并檢測(cè)了細(xì)胞活力。僅siPOOLs產(chǎn)生了較好的一致性表型。

siTOOLs生物技術(shù)始終致力于幫助學(xué)術(shù)研究人員、科學(xué)家、制藥/生物技術(shù)公司和RNAi篩選人員。目前，已幫助許多論文 發(fā)表在《自然》、《細(xì)胞》、《自然醫(yī)學(xué)》、《科學(xué)報(bào)告》等頂級(jí)期刊上。期待與您的合作，我們將為您的RNA研究提供量身定制 的分子工具。

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