miRNeasy Serum/Plasma Kit – microRNA Isolation

用于从动物及人体血浆和血清中纯化包括miRNA的无细胞总RNA

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miRNeasy Serum/Plasma Kit (50)

Cat. No. / ID: 217184

For 50 total RNA preps: 50 RNeasy MinElute Spin Columns, Collection Tubes (1.5 ml and 2 ml), QIAzol Lysis Reagent, RNase-free Reagents and Buffers

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miRNeasy Serum/Plasma Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

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特点

内参用于标准化
洗脱体积最小化
高纯度RNA，适合多种下游应用
简单、灵敏的操作流程
自动化实验方案

产品详情

miRNeasy Serum/Plasma Kit包括检测miRNeasy Serum/Plasma Spike-In Control的miScript Primer Assay，提供一个方便的系统用于标准化。miRNeasy Serum/Plasma Spike-In Control（需单独订购）是C. elegans miR-39 miRNA的类似物，以每管10 pmol冻干粉形式提供。

绩效

miRNeasy Serum/Plasma Kit可从至多200 µl血清或血浆中有效纯化RNA。miRNeasy Kits制备的RNA纯度高，可直接用于敏感的下游应用。miRNeasy操作流程将盐或苯酚污染的可能性降到最低，这些污染可能影响后续分析。

原理

目前miRNA研究中最令人兴奋的领域之一就是对血清或血浆样本中存在的miRNA进行检测。在血清和血浆中存在的相对稳定的胞外miRNA引起了极大兴趣，因为miRNA水平变化可作为潜在的多种疾病的无创性生物标志物。通过从血清或血浆纯化RNA，发现某些癌症出现几种miRNAs的独特表达。miRNeasy Serum/Plasma Kit可从小体积血浆或血清中纯化RNA。血清和血浆中的RNA通常由小于100个核苷酸的分子组成。纯化的RNA可使用miScript PCR System进行生物标志物研究。

miRNeasy Serum/Plasma Kit整合了基于苯酚/胍的样本裂解方法和基于硅胶膜的总RNA纯化方法。试剂盒中包括的QIAzol Lysis Reagent是苯酚和异硫氰酸胍的单相溶液，便于裂解组织，抑制RNases，并去除大部分有机抽提裂解液中的胞内DNA和蛋白。

标准化内参的使用

在血清或血浆样本中miRNA表达谱使用什么作为内参，目前在研究学者间还没有明确共识。很多研究者选择将合成的miRNA导入RNA样品中，以帮助监测RNA回收率和反转效率。QIAGEN推荐miRNeasy Serum/Plasma Spike-In Control作为血清或血浆miRNA表达谱的内参。miRNeasy Serum/Plasma Spike-In Control是C. elegans miR-39 miRNA类似物，使用miScript PCR System配合Ce_miR-39_1 miScript Primer Assay（miRNeasy Serum/Plasma Kit中提供）通过RT-PCR可轻松检测。miRNeasy Serum/Plasma Spike-In Control需单独订购。试剂盒中提供的Ce_miR-39_1 miScript Primer Assay需与miRNeasy Serum/Plasma Spike-In Control配合使用。获取更多细节，可参考miRNeasy Serum/Plasma Handbook。

程序

血清和血浆样本溶于QIAzol Lysis Reagent中。加入氯仿后，裂解液离心后分成水相和有机相。RNA位于上层水相，DNA位于中间相，蛋白位于下层有机相或中间相。

提取上层水相后，加入乙醇以提供RNA结合的合适条件，RNA分子大于18个核苷酸。样品再加入RNeasy MinElute离心柱，总RNA结合到膜上，苯酚和其他污染物被有效洗去。高品质RNA在小体积不含RNase水中洗脱。

血清和血浆中主要含有小RNAs，因此无需分开纯化小RNA和大RNA组分。

使用miRNeasy Serum/Plasma Spike-In Control进行标准化

使用miRNeasy Serum/Plasma Kit纯化miRNA时，加入QIAzol Lysis Reagent后，在加入氯仿分相前，向样本中加入miRNeasy Serum/Plasma Spike-In Control。real-time RT-PCR后，合成miRNA的C_T值可对样本进行标准化，可作为不同RNA纯化产量和扩增效率的对照。此外，通过与合成miRNA的标准曲线C_T值比较，可评估RNA的回收，因为合成miRNA与RNA纯化流程无关。

在QIAcube全自动核酸纯化仪上进行miRNA自动纯化

在QIAcube全自动核酸纯化仪上可完全自动纯化包括miRNA的总RNA。新型QIAcube全自动核酸纯化仪采用先进技术处理QIAGEN离心柱，将自动化、低通量样品制备无缝整合到实验室工作流程中。使用QIAcube全自动核酸纯化仪的样品制备流程与手工操作相同（如裂解、结合、洗涤和洗脱），可使用miRNeasy Serum/Plasma Kit进一步纯化高品质RNA。

应用

miRNeasy Serum/Plasma Kit可纯化包括miRNA的总RNA，用于多种应用，包括：

生物标志物研究

Northern印迹分析

使用miScript PCR System进行定量real-time RT-PCR

微阵列分析

资源

Download Safety Data Sheets for QIAGEN product components.

Poster for download

Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow

FAQ

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro.

Short-term storage (up to 4 weeks) at room temperature (15–25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

FAQ ID - 3560

The miRNeasy Serum/Plasma Spike-In Control (219610) is ordered separately from the miRNeasy Serum/Plasma Kit. It is 10 pmol lyophilized C. elegans miR-39 miRNA mimic.

FAQ ID -3169

It is bacterial ribosomal RNA (Roche), cat. no. 10 206 938 001.

FAQ ID -3173

Yes, please follow the User-Developed Protocol 'Isolation of genomic DNA and/or proteins from fatty tissue samples treated with QIAzol Lysis Reagent' (RY16).

FAQ ID -955

RNA preparations containing miRNA can be cleaned up by modifying* the cleanup protocols listed in the handbooks of the RNeasy Mini Kit or the RNeasy MinElute Cleanup Kit .

* Modify the cleanup protocol at step 2, by increasing the volume of ethanol (96-100%) from 250 µl to 950 µl.

FAQ ID -3002

The miRNeasy Serum/Plasma Spike-in Control (cat n° 219610) will not pose a problem for NGS sequencing. In case of a library prep for larger RNAs, a size selection step may be part of the procedure that removes small RNA in that size range.

Yields of total RNA achieved with the miRNeasy Serum/Plasma Kit vary strongly between samples from different individuals. However, yield of RNA from 50-200 µl human plasma and serum is not quantifiable with photometric methods like NanoDrop; they are usually too low for quantification by OD measurement.

You may use more sensitive colorimetric or fluorimetric assays for quantification, i.e. RiboGreen assays.

Use of miRNeasy Serum/Plasma Spike-In Control (cat. no. 219610) and corresponding Ce_miR-39_1 miScript Primer Assay (cat.no. MS00019789) is recommended to monitor miRNA purification and amplification.

FAQ ID -3171

Both ends of the miRNeasy Serum/Plasma Spike-in Control carry only the hydroxyl groups. The 5'-phosphate is not relevant for the biological function.