QIAamp Circulating Nucleic Acid Kit
For isolation of free-circulating DNA and RNA from human plasma or serum
- Concentration of nucleic acids, with high input and low elution volumes
- Efficient recovery of fragmented DNA and RNA
- No organic extraction or ethanol precipitation
- Removal of contaminants and inhibitors
The QIAamp Circulating Nucleic Acid Kit greatly simplifies concentration and purification of free-circulating DNA and RNA from plasma or serum. The kit can be automated on the QIAcube Connect.
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QIAamp Circulating Nucleic Acid Kit (50)
For 50 preps: QIAamp Mini Columns, Tube Extenders (20 ml), QIAGEN Proteinase K, Carrier RNA, Buffers, VacConnectors, and Collection Tubes (1.5 ml and 2 ml)
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55114
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Carrier RNA(poly A) 12x1350µg-15-25°C,KG
Carrier RNA(poly A) 12x1350µg-15-25°C,KG
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1017647
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QIAamp Circulating Nucleic Acid 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。
回收游离DNA。|回收片段化DNA,表现更出色。|高效纯化游离miRNA。|高效回收甲基化DNA。|
在24个血浆样本中加入同等量的DNA片段(200 bp和1000 bp)。使用QIAamp Circulating Nucleic Acid Kit从5 ml血浆中纯化DNA,洗脱体积为80 μl。使用QuantiTect Multiplex PCR Kit对每个DNA片段特异性的66 bp靶点进行双重real-time PCR,定量DNA的产量。|使用QIAamp Circulating Nucleic Acid Kit从5 ml血浆中纯化游离DNA,或者使用QIAamp MinElute Virus Vacuum Kit从200 μl血浆中纯化游离DNA,洗脱体积为100 μl。使用QuantTect Multiplex PCR Kit对18S rRNA基因内的66 bp扩增片段和500 bp扩增片段进行双重real-time PCR,定量DNA的产量。500 bp扩增片段和66 bp扩增片段的扩增差异显示DNA被片段化,从而导致完整的500 bp目标序列的丰度低于较短的66 bp靶点。(请注意差异大于五倍样本输入体积差异)。|使用QIAamp Circulating Nucleic Acid Kit从4 ml采集血浆中纯化游离DNA,洗脱体积为50 μl。将标准试剂盒实验方案与适用于miRNA的专用实验方案进行比较。使用TaqMan microRNA Assay(Applied Biosystems)定量miRNA 30b的产量,使用Human miScript Assay(QIAGEN)定量miRNA 16的产量。采用miRNA实验方案获得的CT值较低,表示miRNA的产量更高。|使用QIAamp Circulating Nucleic Acid Kit从4 ml血浆中纯化加入的甲基化DNA和非目标poly-A RNA,洗脱体积为45 μl。使用EpiTect Bisulfite Kit(QIAGEN)进行亚硫酸氢盐转化,使用real-time MSP分析和QuantiTect Multiplex PCR Kit组分在ABI PRISM 7900HT Sequence Detection System上进行甲基化特异性PCR(MSP)。对两个全甲基化位点数据进行分析。|
性能
Analysis of tumor-specific extracellular DNA fragments and mRNAs in the blood can enable specific detection of tumor types from a simple blood sample. These circulating nucleic acids are present in serum or plasma usually as short fragments, <1000 bp (DNA) or <1000 nt (RNA). In addition, miRNAs, as small as 21 nt, have the potential to provide biomarkers for certain cancers and disease states.
The QIAamp Circulating Nucleic Acid Kit enables efficient purification of these circulating nucleic acids from human plasma or serum and other cell-free body fluids. Efficient purification with reproducible yields provides a representative population of the circulating nucleic acids in blood (see figure "Reproducible recovery of circulating DNA"). Tube extenders and vacuum processing on the QIAvac 24 Plus enable starting sample volumes of up to 5 ml, and flexible elution volumes between 20 μl and 150 μl allow concentration of nucleic acid species that are present in low concentrations. The kit provides advanced technology of selective binding to a silica-based membrane for improved recovery of fragmented nucleic acids (see figure "Improved recovery of fragmented DNA").
Purification of circulating RNA, without copurification of DNA, is possible with DNA digestion using the RNase-Free DNase Set. A specialized protocol provides highly efficient purification of small RNA, such as miRNAs (see figure "Efficient purification of circulating miRNA"). Methylated DNA can be efficiently purified using the QIAamp Circulating Nucleic Acid Kit. The purified DNA maintains its methylation, allowing bisulfite conversion for analysis of the methylation state (see figure "Efficient recovery of methylated DNA").
The purified and concentrated circulating DNA and RNA is free of proteins, nucleases, and other impurities, and is ready to use in wide range of downstream applications, including:
- PCR and quantitative real-time PCR and RT-PCR
- Biomarker research and validation for blood-based cancer detection
- Viral nucleic acid detection
原理
The QIAamp Circulating Nucleic Acid Kit simplifies isolation of circulating DNA and RNA from plasma or serum. No phenol–chloroform extraction is required. Nucleic acids bind specifically to the QIAamp Mini column, while contaminants pass through. PCR inhibitors, such as divalent cations and proteins, are completely removed in 3 wash steps, leaving pure nucleic acids to be eluted in a buffer provided with the kit. The QIAamp Circulating Nucleic Acid technology yields circulating DNA and RNA from human plasma, serum, or urine. The QIAamp Circulating Nucleic Acid Kit combines the selective binding properties of silica-based membrane with flexible elution volumes between 20 and 150 μl. Circulating RNA can be purified with DNA digestion using the RNase-Free DNase Set.
操作流程
The QIAamp Circulating Nucleic Acid procedure includes 4 steps (lyse, bind, wash, and elute) that are carried out using QIAamp Mini columns on a vacuum manifold. The simple procedure is highly suited for simultaneous processing of multiple samples, providing nucleic acids in less than 2 hours per 24 samples. If the QIAamp Circulating Nucleic Acid Kit is used for isolation of viral RNA and DNA, the performance cannot be guaranteed for every virus species and must be validated by the user.
应用
The QIAamp Circulating Nucleic Acid Kit efficiently purifies and concentrates free-circulating DNA, RNA, miRNA, and viral nucleic acids from starting materials that contain low concentrations of mostly fragmented DNA and RNA (typically 1–100 ng/ml circulating DNA in human plasma). Starting sample volumes can be up to 5 ml. The QIAamp Circulating Nucleic Acid Kit purifies and concentrates nucleic acids from the following sample types:
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特点
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参数
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应用
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PCR, real-time PCR
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符合CE/FDA/IVD
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洗脱体积
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20–150 µl
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规格
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QIAamp Mini Columns
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主要样本类型
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Serum, plasma
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处理
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Manual (vacuum)
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纯化总RNA、miRNA、poly A+ mRNA、DNA或蛋白
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Free-circulating DNA, RNA and miRNA, viral DNA, viral RNA
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样本量
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1-5 ml
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技术
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Silica technology
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每次运行或制备时间
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<2 hour for 24 preps
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产量
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Varies, due to donor-to-donor variations and disease status
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FAQ ID -635
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浏览
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FAQ ID -728
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浏览
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For concentration and purification of free-circulating DNA and RNA from human plasma or serum
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图片
回收游离DNA。
在24个血浆样本中加入同等量的DNA片段(200 bp和1000 bp)。使用QIAamp Circulating Nucleic Acid Kit从5 ml血浆中纯化DNA,洗脱体积为80 μl。使用QuantiTect Multiplex PCR Kit对每个DNA片段特异性的66 bp靶点进行双重real-time PCR,定量DNA的产量。
回收片段化DNA,表现更出色。
使用QIAamp Circulating Nucleic Acid Kit从5 ml血浆中纯化游离DNA,或者使用QIAamp MinElute Virus Vacuum Kit从200 μl血浆中纯化游离DNA,洗脱体积为100 μl。使用QuantTect Multiplex PCR Kit对18S rRNA基因内的66 bp扩增片段和500 bp扩增片段进行双重real-time PCR,定量DNA的产量。500 bp扩增片段和66 bp扩增片段的扩增差异显示DNA被片段化,从而导致完整的500 bp目标序列的丰度低于较短的66 bp靶点。(请注意差异大于五倍样本输入体积差异)。
高效纯化游离miRNA。
使用QIAamp Circulating Nucleic Acid Kit从4 ml采集血浆中纯化游离DNA,洗脱体积为50 μl。将标准试剂盒实验方案与适用于miRNA的专用实验方案进行比较。使用TaqMan microRNA Assay(Applied Biosystems)定量miRNA 30b的产量,使用Human miScript Assay(QIAGEN)定量miRNA 16的产量。采用miRNA实验方案获得的CT值较低,表示miRNA的产量更高。
高效回收甲基化DNA。
使用QIAamp Circulating Nucleic Acid Kit从4 ml血浆中纯化加入的甲基化DNA和非目标poly-A RNA,洗脱体积为45 μl。使用EpiTect Bisulfite Kit(QIAGEN)进行亚硫酸氢盐转化,使用real-time MSP分析和QuantiTect Multiplex PCR Kit组分在ABI PRISM 7900HT Sequence Detection System上进行甲基化特异性PCR(MSP)。对两个全甲基化位点数据进行分析。
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