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RNeasy Midi Kit

从细胞、组织或酵母中纯化至多1 mg总RNA
货号：75144
品牌：QIAGEN
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特点

  •     · 数分钟内快速纯化得到高品质总RNA
  •     · 即用型RNA在各种下游应用中表现良好
  •     · 大量起始样本，RNA产量稳定
  •     · 无需酚/氯仿抽提
  •     · 无需CsCl梯度离心，无LiCl或者乙醇沉淀

 

产品详情

RNeasy Kits 是总RNA分离的金标准。它们使用硅胶膜RNeasy spin columns 或96孔板从少量到大量细胞、组织和酵母中快速纯化高质量RNA。使用RNAprotect Tissue Reagent 或 Allprotect Tissue Reagent 可以方便地稳定组织样品，并使用TissueRuptor II或TissueLyser II或LT系统有效破坏组织样品。RNeasy 96 Kit能够使用硅膜RNeasy 96板从多达96个培养细胞样品中高通量纯化总RNA。专用RNeasy QIAcube Kit 可在QiaCube Connect上自动纯化1-12个样品。

 

性能

RNeasy Kits deliver highly reproducible yields of total RNA from small to large samples. Total RNA can reliably be purified from small numbers of cells, including a single cell, as well as from small amounts of standard tissues (参见 

Reliable RNA isolation from a single cell.

Total RNA was isolated from the indicated number of HeLa cells using the RNeasy Micro Kit. [A] Amplification plot. [B] Correlation of crossing point (CP) values and cell numbers. Real-time RT-PCR was carried out on the LightCycler System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for β-actin.
"> "Reliable RNA isolation from a single cell", 

Highly reproducible yields for sensitive applications.

Total RNA was isolated by 5 different users in duplicate from 2 mg of the indicated rat tissues, using the RNeasy Micro Kit. Quantitative, real-time RT-PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for c-jun.

"> "Highly reproducible yields for sensitive applications

Highly reproducible yields for sensitive applications.

Total RNA was isolated by 5 different users in duplicate from 2 mg of the indicated rat tissues, using the RNeasy Micro Kit. Quantitative, real-time RT-PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe RT-PCR Kit, with primers and probes specific for c-jun.

">" and  "High-quality total RNA from fine needle aspirates") using the RNeasy Micro Kit. RNA purified using the RNeasy Micro Kit provides maximum sensitivity with precious samples in quantitative gene expression analyses, such as real-time RT-PCR, by efficient on-column digestion of genomic DNA (参见 

Efficient on-column removal of genomic DNA.

Total RNA was isolated from the indicated number of HeLa cells using the RNeasy Micro Kit. Quantitative, real-time PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe PCR Kit, with primers and probes specific for c-jun. In order to evaluate the efficiency of the on-column DNase digestion, each sample was amplified after reverse transcription (+RT) and directly, without a reverse transcription step (-RT). The lack of signal in the "-RT" samples indicates that no DNA was detectable. Of the "-RT" results, only the 50,000 cell data points are visible.
"> "Efficient on-column removal of genomic DNA

Efficient on-column removal of genomic DNA.

Total RNA was isolated from the indicated number of HeLa cells using the RNeasy Micro Kit. Quantitative, real-time PCR was carried out on an ABI Sequence Detection System using the QuantiTect Probe PCR Kit, with primers and probes specific for c-jun. In order to evaluate the efficiency of the on-column DNase digestion, each sample was amplified after reverse transcription (+RT) and directly, without a reverse transcription step (-RT). The lack of signal in the "-RT" samples indicates that no DNA was detectable. Of the "-RT" results, only the 50,000 cell data points are visible.
">").
Total RNA is easily purified from animal or human cells, animal or human tissues, and yeast (see table “Total RNA yields obtained with RNeasy Kits” and figure 

RT-PCR of RNA from as few as 100 cells.

RT-PCR of total RNA isolated with the RNeasy Mini Kit from the indicated numbers of HeLa cells. 10 µl (1/5) of eluate was digested with RNase-free DNase and reverse transcribed with oligo-dT primer. 2.5 µl (1/20) of the cDNA mix was used in 50 µl PCR. A 452 bp fragment of GAPDH was amplified. C-: negative control; C+: positive control; M: 100 bp ladder.
"> "RT-PCR of RNA from as few as 100 cells").

 

使用RNeasy Kits 获得的总RNA产量

Source Starting material Average yield
Animal cells Micro Mini Midi Maxi 96-well Micro Mini Midi Maxi c
LMH 5 x 105 1 x 106 7 x 107 5 x 108 1 x 105 – 12 µg 850 µg 5.7 mg 1.3 µg*
HeLa 5 x 105 1 x 106 7 x 107 4 x 108 1 x 105 5mg 15 µg 1000 µg 6.0 mg 1.6 µg*
COS-7 5 x 105 1 x 106 3 x 107 1.8 x 108 1 x 105 17.5 mg 35 µg 950 µg 5.8 mg 3.1 µg*
Lymphocytes
(unstimulated)		 5 x 105 1 x 106 1 x 108 5 x 108 – – 0.5 µg 50 µg 0.3 mg –
Huh7 5 x 105 – – – 1 x 105 7.5mg – – – 2.0 µg*
Jurkat 5 x 105 – – – 1 x 105 – – – – 1.4 µg*
Mouse tissue
Liver 5 mg 10 mg 200 mg 1 g – 15 µg 40 µg 700 µg 3.6 mg –
Lung 5 mg 10 mg 100 mg 0.5 g – 5 µg 10 µg 130 µg 0.6 mg –
Spleen 5 mg 10 mg 200 mg 1 g – 15 µg 35 µg 600 µg 3.2 mg –
Yeast cells
S. cerevisiae – 1 x 107 2 x 108 1 x 109 – – 25 µg 450 µg 2.4 mg –

*数量可能因发育阶段、使用的生长条件等而异。

 

The dedicated RNeasy Mini QIAcube Kit, including rotor adapters preloaded with RNeasy spin columns and elution tubes, delivers greater convenience and time savings (参见  "Significant time savings with dedicated QIAcube Kits").

Total RNA purified with the RNeasy Maxi Kit is of high quality and is suitable for many downstream applications (参见  "High-quality RNA from a variety of samples"). Total RNA is easily purified with the RNeasy Maxi Kit from large amounts of starting material including animal or human cells, animal or human tissues, and yeast cells (see table “Total RNA yields obtained with RNeasy Kits”).

The RNeasy 96 system provides fast and reproducible total RNA purification for high-throughput gene expression profiling. The RNA is suitable for sensitive applications such as quantitative, real-time RT-PCR and microarray analysis (参见 High-quality RNA for sensitive analysis of a low-copy transcript."> "High-quality RNA for sensitive analysis of a low-copy transcript"). Sample sizes range from 10 to 5 x 105 cells (see figure  "RT-PCR of RNA from as few as 100 cells"), and high-quality RNA can be purified from large numbers of samples (参见 Reproducible yields of high-quality RNA."> "Reproducible yields of high-quality RNAReproducible yields of high-quality RNA.">"). Individual variations are low throughout the entire purification process; TaqMan® threshold-cycle values are easily achieved at the end of the process with a coefficient of variation (CV) less than 3% using the QuantiTect Probe RT-PCR Kit (参见 Repeatability of automated RNA purification."> "Repeatability of fully automated RNA purification").

 

原理

RNeasy Kits allow efficient purification of total RNA from small to large amounts of starting material, including samples such as microdissected tissues and fine needle aspirates, from milligram amounts of fibrous tissues including heart and muscle tissue, and from small numbers of cells down to single cells (e.g., FACS sorted cells). For microdissected FFPE tissues, we recommend the RNeasy FFPE Kit. RNeasy technology simplifies total RNA isolation from cells, tissues and yeast by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. RNeasy Kits provide the highest-quality RNA with minimum copurification of DNA.

 

步骤

RNeasy technology simplifies total RNA isolation (参见 “Amount of starting material for RNeasy procedures”). Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. For certain RNA applications that are sensitive to very small amounts of DNA, the residual amounts of DNA remaining can be removed using a convenient on-column DNase treatment. Pure, concentrated RNA is eluted in water. A variety of special application protocols is also available.

Automated processing on the QIAcube Connect

The QIAcube Connect uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into laboratory workflows. All steps in the purification procedure are fully automated — and up to 12 samples can be processed per run. The QIAcube Connect together with the dedicated RNeasy Mini QIAcube Kit provides fast, easy, and convenient RNA purification.
 
High-throughput processing in 96-well format

The RNeasy 96 system provides a fast and efficient procedure. Cells can be grown and directly lysed in 96-well cell-culture dishes. After transfer of the lysates to the wells of the RNeasy 96 plate, RNA binds to the silica membrane in each well, and contaminants are washed away. Pure RNA is then eluted in RNase-free water into individual collection tubes suitable for long-term storage and is ready to use for any experiment. The RNeasy 96 Kit includes protocols for total RNA isolation, cytoplasmic RNA isolation, and RNA cleanup. RNeasy 96 plates can be processed on the QIAvac 96 vacuum manifold and/or the 96-Well-Plate Centrifuge System.

 

RNeasy程序的起始材料量

Amount of
starting material		 RNeasy Micro Kit RNeasy Mini Kit RNeasy Mini QIAcube Kit RNeasy Midi Kit RNeasy Maxi Kit RNeasy 96 Kit
Cells <5 x 105 cells 10 to 1 x 107 animal or human cells 1 x 107 cells 5 x 106 to 1 x 108 animal or human cells Up to 5 x 108 animal or human cells 10–5 x 105 cells
Tissue <5 mg tissue 0.5–30 mg animal or human tissues 30 mg tissue 20–250 mg animal or human tissues 150 mg to 1 g tissue –
Yeast   <5 x 107 yeast cells – 2 x 107 to 5 x 108 yeast cells 2.5 x 109 yeast cells –

 

 

应用

使用RNeasy技术纯化的RNA的A260/280比率为1.9–2.1（以10mM Tris·Cl、pH 7.5测量），非常适合用于所有应用。下游应用包括：

    · 转录组测序

    · 定量实时RT-PCR

    · 从一个细胞开始的实时RT-PCR

    · 终点RT-PCR

    · Northern、点和槽印迹法

    · 阵列分析

    · Poly A+RNA选择

 

RNeasy试剂盒的比较

Features RNeasy Micro Kit RNeasy Mini Kit RNeasy Mini QIAcube Kit RNeasy Midi Kit RNeasy Maxi Kit RNeasy 96 Kit
Applications RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis		 RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis		 RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis		 RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis		 RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis		 RNA-seq, quantitative real-time RT-PCR,
end-point RT-PCR, Northern dot and slot blotting,
Microarray analysis
Elution volume 10–14 µl 30–100 µl 30–100 µl 300–500 µl 800–2400 µl 45–140 µl
Format Spin column Spin column Spin column, preloaded into rotor adapters Spin column Spin column 96-well plate
Main sample type Tissue, cells Tissue, cells, yeast Tissue, cells, yeast Tissue, cells, yeast Tissue, cells, yeast Cultured cells
Processing Manual (centrifugation) Manual (centrifugation) Automated (QIAcube Connect) Manual (centrifugation) Manual (centrifugation) Manual (centrifugation or vacuum)

Purification of total RNA, miRNA,
poly A+ mRNA, DNA or protein

 Total RNA Total RNA Total RNA Total RNA Total RNA Total RNA
Sample amount <5 mg tissue,
<5 x 105 cells		 0.5–30 mg tissue,
10 to 1 x 107 animal or human cells		 0.5–30 mg tissue,
1 x 107 cells		 20–250 mg tissue,
5 x 106 to 1 x 108 animal or human cells		 150 mg – 1 g tissue,
Up to 5 x 108 animal or human cells		 5 x 105
Technology Silica technology Silica technology Silica technology Silica technology Silica technology Silica technology
Time per run or per prep 30–40 minutes 20 minutes – <1 hour 1 hour 30–40 minutes
Yield Varies Varies Varies Varies Varies 1.3–3.1 µg

 

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