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RNeasy Protect Bacteria Mini Kit

从细菌中稳定并纯化至多100 μg的总RNA

  • 立即稳定和保护体内的RNA
  • 基因表达和基因谱数据可靠
  • 无需使用液氮、干冰或苯酚
  • 快速获得即用型高品质总RNA
  • 无需氯化铯梯度离心,无需氯化锂或乙醇沉淀

RNeasy Protect Bacteria Mini Kit包含RNAprotect Bacteria Reagent和RNeasy离心柱,前者用于稳定细菌样本中的RNA,后者使用硅胶膜技术纯化得到至多100 µg高品质RNA。可通过使用TissueLyser II实现高效破碎细菌样品。纯化过程可在QIAcube全自动核酸纯化仪上自动运行。

Cat No./ID: 74524
RNeasy Protect Bacteria Mini Kit (50)
RNeasy Mini Kit (50) and RNAprotect Bacteria Reagent (2 x 100 ml)

RNeasy Protect Bacteria Mini Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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Accurate gene expression profiles.
Total RNA was isolated from RNAprotect stabilized E. coli cultures using the RNeasy Protect Bacteria Kit (RNeasy Protect Bacteria) or from unstabilized cultures using a commercial precipitation method (Supplier EIII). To distinguish between gene expression under defined culture conditions and effects of artifactual gene induction during harvesting and RNA isolation, the RNA polymerase inhibitor rifampicin was added to half of the culture prior to RNA isolation. Differences in transcript levels with and without rifampicin therefore generally reflect the degree of RNA degradation. [A] GeneChip analysis of E.coli microarrays was carried out according to standard Affymetrix protocols. [B] The percentage of genes expressed was estimated as the number of different transcripts determined present by GeneChip analysis divided by the total number of transcripts represented on the microarray. (Data from a collaborative study with Affymetrix.)
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RNAprotect Bacteria Reagent procedure.
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RNAprotect Bacteria Reagent prevents mRNA degradation.
In order to monitor mRNA degradation only, transcription was stopped by adding the RNA polymerase inhibitor rifampicin to a growing culture of E. coli. The culture was split into halves, and RNAprotect Bacteria Reagent was added to one half. Samples were left at room temperature for 0, 4, 8, and 16 minutes before centrifugation and RNA isolation. The resulting RNA was analyzed by agarose gel electrophoresis (top panel). The expression of two marker genes with different half-lives was examined by northern blot analysis. Middle panel: ompA (half life of 15 minutes); bottom panel: α-lactamase (half life of 2–5 minutes).
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RNeasy Mini离心柱。
RNeasy Protect Bacteria Mini Kit中包含RNeasy Mini离心柱。
Performance
使用传统方法进行细胞收集和RNA分离时,RNA酶解会导致很多转录体的减少或丢失。这种减少在细菌mRNA分子中尤其明显,因为它们通常只有几分钟的半衰期。此外,在操作处理细菌样本过程中,一些基因会被诱导,导致部分基因的高表达。使用RNAprotect Bacteria Reagent可以在分离RNA之前迅速稳定RNA(参见"RNAprotect Bacteria Reagent prevents mRNA degradation"和"Accurate gene expression profiles")。E. coli(6 x 108个细胞)的RNA产量是100 µg,Bacillus subtilis(1 x 108个细胞) 的RNA产量是15 µg。用此试剂盒纯化得到的RNA品质高,其A260/A280比值为1.9–2.1(测于10 mM Tris·Cl、pH 7.5)。
Principle
RNeasy Protect Bacteria Mini Kit提供RNA保护和分离的完整解决方案,在一个试剂盒中完成从样品收集到RNA纯化的整个过程。经验证的RNeasy硅胶膜技术与用于稳定RNA的RNAprotect Bacteria Reagent相结合,可纯化得到高品质、完整的RNA。这个过程有效地保护了样品收集过程中的细菌表达谱,确保可靠的基因表达分析结果。样本稳定后,RNeasy技术整合了异硫氰酸胍裂解的严谨性和硅胶膜纯化的纯度及速度,过程更加简洁(参见"RNeasy Mini spin column")。
Procedure

在RNA分离前,直接向1体积的细菌培养基(≤7.5 x 108个细菌)中加入2体积的RNAprotect Bacteria Reagent,可立即稳定RNA(参见"RNAprotect Bacteria Reagent procedure")。对样本的稳定使细菌的高效裂解有多种可选的实验方案:酶裂解、机械破碎或两种方法结合使用。我们推荐使用TissueLyser II进行高效的机械破碎。然后将乙醇加入裂解液中以提供理想的结合条件。裂解液加到RNeasy硅胶膜上(结合能力达100 µg RNA)。RNA结合到膜上后,所有污染物被高效洗去。可用30–100 µl水洗脱得到浓缩的纯RNA。RNeasy Protect Bacteria Mini Kit可在QIAcube全自动核酸纯化仪上实现自动化。

由于物种和生长状况不同可能导致从细菌中分离的RNA产量不同。RNeasy Protect Bacteria Mini Kit适用于各种细菌,包括革兰氏阳性菌(如Staphylococcus aureusMycobacterium avium)和革兰氏阴性菌(如Escherichia coliSalmonella typhimurium)。生长在限制或完全培养基中的细菌均可使用。由于RNeasy操作流程能纯化大于200 nt的RNA,得到的RNA中不包含5S rRNA、tRNAs和其他低分子量RNAs。RNeasy Protect Kits提供最高品质RNA,几乎无DNA残留。对微量DNA非常敏感的RNA应用,残留的DNA可在RNeasy操作流程中使用RNase-Free DNase Set方便地进行DNase柱上处理。

Applications

使用RNeasy Protect Bacteria Mini Kit纯化的RNA非常适用于多种应用。下游应用包括:

Northern印迹、点印迹和狭缝印迹
终点法RT-PCR
定量real-time RT-PCR
芯片分析
Features
Specifications
应用 PCR, qPCR, real-time RT-PCR, microarray
洗脱体积 30–100 µl
规格 Spin column
主要样本类型 Bacteria
处理 Manual (centrifugation)
纯化总RNA、miRNA、poly A+ mRNA、DNA或蛋白 RNA
样本量 15 –100 µg
稳定 Yes
技术 Silica technology
每次运行或制备时间 20 minutes
产量 8–70 µg

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试剂盒操作手册 (1)
RNAprotect Bacteria ReagentFor in vivo stabilization of total RNA in bacteriaRNeasy Protect Bacteria Mini KitRNeasy Protect Bacteria Midi KitFor in vivo stabilization of total RNA in bacteria and subsequent RNA purification
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参考文献
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