地高辛标记的Northern blot检测鼠疫菌sRNA

Detection of Yersinia pestis sRNA by digoxigenin-labeled Northern blot

摘要:

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0.005 μg和 0.05 μg。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。

关键词:关键词:sRNA，RNA 寡核苷酸探针，地高辛，Northern blot

Abstract:

Abstract:［Objective］With the application of high-throughput sequencing methods，more and more sRNAs are required to be verified． In this study，we developed the digoxigenin-labeled Northern blot method for detection of Yersinia pestis sRNA．［Methods］Total RNAs extracted from Yersinia． pestis grown under low-iron conditions were loaded onto 10% denaturing urea polyacrylamide gel (dPAGE)，electrophoresed and transferred to nylon membranes． Northern blots were fixed to the membrane by UV cross-linking and subjected to hybridization with 3’-end digoxigenin-labeled oligonucleotides RNA probe for RyhB1 and RyhB2 overnight and followed by washing，blocking，immunological detection and finally exposed to film． ［Results］Exposure time of digoxigenin-labelled Northern blot was 20s-3min． The detection sensitivity of RyhB1 and RyhB2 was 0.005μg and 0.05μg，respectively． RyhB1 and RyhB2 probe specificity was high and no cross reaction with each other was found． Positively charged or neutral nylon membranes are applicable to the hybridization reaction． Hybridization for RNA probe can proceed within 42 to 65℃ with reduced non-specific probe interactions by increasing the temperature while the hybridization temperature for DNA oligonucleotide probes seemed to be determined empirically．[Conlusion］A digoxigenin-labeled Northern blot has been developed for detection of Yersinia pestis sRNA，which was characterized by good specificity，high sensitivity，longer shelf life and short exposure times and provided an power tool for validation of bacterial sRNAs and function studies．

Key words:Keywords: sRNA，RNA oligonucleotide probe，DIG，Northern blot