【摘要】：【目的】隨著高通量測(cè)序方法的應(yīng)用,越來(lái)越多的sRNA(small non-coding RNA,sRNA)需驗(yàn)證。本研究建立用地高辛標(biāo)記Northern blot檢測(cè)鼠疫菌sRNA的技術(shù),為細(xì)菌sRNA驗(yàn)證提供一種靈敏、特異的方法�！痉椒ā吭诘丸F條件下,提取鼠疫菌總RNA,10%dPAGE分離后電轉(zhuǎn)到尼龍膜上并用紫外線交聯(lián)RNA。膜經(jīng)地高辛標(biāo)記RyhB1或RyhB2寡核苷酸RNA探針過(guò)夜雜交后洗脫、封閉和免疫檢測(cè),最后曝光顯影�！窘Y(jié)果】地高辛標(biāo)記的Northern blot曝光時(shí)間為20 s-3 min,RyhB1或RyhB2檢測(cè)靈敏度分別為0.005μg和0.05μg。RyhB1或RyhB2探針特異性好,相互間無(wú)交叉反應(yīng)。帶正電或中性的尼龍膜都適用于雜交反應(yīng)。RNA探針在42℃-65℃內(nèi)雜交均可,提高溫度可減少非特異性反應(yīng);而DNA探針雜交溫度需摸索�！窘Y(jié)論】本研究成功構(gòu)建一種地高辛標(biāo)記Northern blot檢測(cè)鼠疫菌sRNA技術(shù),具有特異性好、靈敏度高、探針易保存、曝光時(shí)間短等優(yōu)點(diǎn),為細(xì)菌sRNA驗(yàn)證和功能研究提供有利工具。
[Abstract]:[objective] with the application of high-throughput sequencing, more and more sRNA (small non-coding RNA,sRNA need to be verified. In this study, a technique for detection of Yersinia pestis sRNA by digoxigenin labeled Northern blot was established, which provided a sensitive and specific method for the verification of bacterial sRNA. [methods] Total RNA, of Yersinia pestis was extracted under low iron conditions. Electroporation of 10%dPAGE onto nylon membrane and UV crosslinking of RNA. After overnight hybridization with digoxin labeled RyhB1 or RyhB2 oligonucleotide RNA probes, the membrane was eluted, blocked and detected by immunoassay. [results] the exposure time of Northern blot labeled with digoxin was 20 s-3 min,. The sensitivity of RyhB1 or RyhB2 was 0.005 渭 g and 0. 05 渭 g.RyhB1 or RyhB2, respectively. Nylon membrane with positive charge or neutral can be used in hybridization reaction. RNA probe can be hybridized within 42 鈩，

本文編號(hào)：2328014