地高辛标记的Northern blot检测鼠疫菌sRNA
发布时间：2018-11-12 19:32
【摘要】：【目的】随着高通量测序方法的应用,越来越多的sRNA(small non-coding RNA,sRNA)需验证。本研究建立用地高辛标记Northern blot检测鼠疫菌sRNA的技术,为细菌sRNA验证提供一种灵敏、特异的方法。【方法】在低铁条件下,提取鼠疫菌总RNA,10%dPAGE分离后电转到尼龙膜上并用紫外线交联RNA。膜经地高辛标记RyhB1或RyhB2寡核苷酸RNA探针过夜杂交后洗脱、封闭和免疫检测,最后曝光显影。【结果】地高辛标记的Northern blot曝光时间为20 s-3 min,RyhB1或RyhB2检测灵敏度分别为0.005μg和0.05μg。RyhB1或RyhB2探针特异性好,相互间无交叉反应。带正电或中性的尼龙膜都适用于杂交反应。RNA探针在42℃-65℃内杂交均可,提高温度可减少非特异性反应;而DNA探针杂交温度需摸索。【结论】本研究成功构建一种地高辛标记Northern blot检测鼠疫菌sRNA技术,具有特异性好、灵敏度高、探针易保存、曝光时间短等优点,为细菌sRNA验证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，
本文编号：2328014
[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，
本文编号：2328014
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