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MiRNA定量PCR|實(shí)驗(yàn)技術(shù)服務(wù)

時(shí)間:2015/6/2閱讀:538
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關(guān)鍵詞:MiRNA定量PCR|實(shí)驗(yàn)技術(shù)服務(wù)
簡(jiǎn)介:世界*品牌MiRNA定量PCR|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
MiRNA定量PCR|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:
實(shí)時(shí)熒光定量PCR
從1996年美國(guó)AppliedBiosystems公司推出實(shí)時(shí)熒光定量PCR技術(shù)以來,該技術(shù)已經(jīng)越來越廣泛的應(yīng)用到各類RNA的研究中,,成為研究RNA*的實(shí)驗(yàn)手段之一,。目前比較常用的方法有兩種:
(1)TaqMan熒光探針法:該方法在特異性探針的兩端分別標(biāo)記一個(gè)報(bào)告熒光基團(tuán)和一個(gè)淬滅熒光基團(tuán)。當(dāng)探針完整時(shí),,熒光基團(tuán)發(fā)光被淬滅基團(tuán)吸收,;在PCR擴(kuò)增過程中,探針被降解,,熒光基團(tuán)與淬滅基團(tuán)分離,,發(fā)出可被檢測(cè)到的熒光,以此來監(jiān)測(cè)整個(gè)PCR過程,。
(2)SYBR熒光染料法:該方法利用了SYBRGreen熒光染料非特異性與雙鏈DNA結(jié)合并發(fā)光的特性,,檢測(cè)PCR的全部進(jìn)程,從而判定初始RNA的量。
常用的miRNA*鏈合成方法
成熟的miRNA大小只有22 nt左右,,在用實(shí)時(shí)熒光定量PCR檢測(cè)miRNA時(shí),,難點(diǎn)在于如何識(shí)別如此小的miRNA并合成*鏈。目前常用的用于識(shí)別并合成miRNA*鏈的方法主要有頸環(huán)法和加尾法兩種,。
由于成熟miRNA較小,,無法用常規(guī)的方法直接進(jìn)行反轉(zhuǎn)錄、PCR,。所以上述兩種方法分別加PolyA尾及頸環(huán)結(jié)構(gòu)的方法,,將miRNA配對(duì)的序列延長(zhǎng),然后進(jìn)行正常的反轉(zhuǎn)錄及后續(xù)的PCR檢測(cè),。頸環(huán)法只針對(duì)成熟miRNA,,特異性相對(duì)較高,但操作繁瑣,,成本較高,;加尾法可以檢測(cè)到成熟miRNA及pre-miRNA,特異性稍低,,但操作及引物設(shè)計(jì)簡(jiǎn)單,。
近年來,研究人員發(fā)現(xiàn)了越來越多的miRNA,,其中有很多miRNA序列相近,,有的僅僅只有一個(gè)堿基差異。這些不同的miRNA雖然序列相近,,但是也有著不同的來源及功能,。上述兩種方法雖然解決了miRNA的識(shí)別及后續(xù)PCR問題,但是無法分辨這些只有一個(gè)或者兩個(gè)堿基差異的miRNA,。美國(guó)Signosis推出了一種新的miRNA識(shí)別方法,,可以分辨僅有單個(gè)堿基差異的miRNA。 
美國(guó)Signosis推出的新方法,,使用多對(duì)寡核苷酸對(duì)目的miRNA進(jìn)行識(shí)別,,單個(gè)堿基差異就可以破壞寡核苷酸與靶標(biāo)序列的結(jié)合,從而分辨單個(gè)堿基差異的miRNA,;同時(shí)該方法不用進(jìn)行方轉(zhuǎn)錄,,避免了反轉(zhuǎn)錄過程中出現(xiàn)錯(cuò)配的可能;同時(shí),,這個(gè)新方法還引入了磁珠分離技術(shù),,在進(jìn)行PCR前,通過結(jié)合有鏈霉親和素的磁珠對(duì)特異性標(biāo)記有生物素的靶標(biāo)序列進(jìn)行純化,,大大提高了特異性,。

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