For purification of up to 100 µg total RNA from cells/tissues using gDNA Eliminator columns

  • Unique gDNA Eliminator columns avoid the need for DNase
  • Efficient removal of genomic DNA
  • Highly reproducible yields of RNA in minutes
  • High-performance RNA for sensitive applications
The RNeasy Plus Mini Kit integrates fast, convenient purification of up to 100 µg RNA with effective elimination of genomic DNA. Cell or tissue lysates are spun through gDNA Eliminator spin columns to remove genomic DNA. Total RNA is then purified using RNeasy Mini spin columns. The kit can be automated on the QIAcube. Tissue samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. For smaller samples, the RNeasy Plus Micro Kit (spin-column binding capacity of 45 µg RNA) is also available.
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Product Cat. no. List price:
RNeasy Plus Mini Kit (50)
For 50 minipreps: RNeasy Mini Spin Columns, gDNA Eliminator Spin Columns, Collection Tubes, RNase-Free Water and Buffers
74134
327,00 €
RNeasy Plus Mini Kit (250)
For 250 minipreps: RNeasy Mini Spin Columns, gDNA Eliminator Spin Columns, Collection Tubes, RNase-Free Water and Buffers
74136
1.439,00 €
The RNeasy Plus Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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High, reproducible RNA yields.|Effective tissue genomic DNA removal.|RNeasy Plus procedure.|Effective cell genomic DNA removal.|Array-ready RNA.|
Total RNA was purified in duplicate from different amounts of Jurkat cells using the RNeasy Plus Mini Kit or a similar kit from Supplier AV. Real-time RT-PCR assays for β-actin were performed (40 cycles). The lower CT values with the RNeasy Kit demonstrate greater RNA yields. With the kit from Supplier AV, no transcript was detectable in RNA purified from 102 cells. |Total RNA was purified in duplicate from various mouse tissues (10 mg per sample) using the RNeasy Plus Mini Kit or kits from other suppliers. Real-time PCR assays for c-jun were performed to determine the amount of DNA contamination in the purified RNA.|A short workflow enables RNA purification with genomic DNA removal in less than 25 minutes.|Total RNA was purified from Jurkat cell samples (1 x 106 cells per sample) using [A] the RNeasy Plus Mini Kit, or [B] an RNA purification kit with integrated genomic DNA removal from Supplier AV. Duplicate real-time RT-PCR assays for β-actin were performed with (+RT) or without (-RT) reverse transcriptase. The -RT curves demonstrate that RNA purified using the RNeasy Plus Mini Kit was virtually free of genomic DNA.|Total RNA was purified in duplicate from 1 x 106 HeLa cells using the RNeasy Plus Mini Kit. cRNA was prepared from the duplicate RNA samples (3.5 μg each) using the GeneChip IVT Labeling Kit. The cRNA samples (15 μg each) were analyzed on GeneChip Human Genome U133A probe arrays. The scatter plot shows the correlation between the two samples (Pearson correlation coefficient [r] is 0.996). Red: gene present in both samples; Blue: gene absent or marginal in one sample; Yellow: gene absent or marginal in both samples.|
Performance
Purification of RNA from cells and tissues with the RNeasy Plus Mini procedure allows high, reproducible RNA yields and effective elimination of genomic DNA contamination for sensitive applications (see figures "Effective cell genomic DNA removal", "Effective tissue genomic DNA removal", "High, reproducible RNA yields", and "Array-ready RNA"). Total RNA with Agilent RIN values of close to 10 is routinely obtained from cultured cells.
Principle

Cells and easy-to-lyse tissues are lysed and homogenized in highly denaturing guanidine-isothiocyanate–containing Buffer RLT Plus, which immediately inactivates RNases to ensure isolation of intact RNA. The lysate is then passed through a gDNA Eliminator spin column. This column, in combination with the high-salt buffer, selectively and efficiently removes genomic DNA. Ethanol is added to provide appropriate binding conditions for RNA, and the sample is applied to an RNeasy MinElute spin column. These specialized columns contain a silica membrane that specifically binds RNA from lysed cells.

Procedure

Total RNA is purified from up to 107 cells or 30 mg tissue. A short workflow enables RNA purification with genomic DNA removal in less than 25 minutes (see flowchart "RNeasy Plus procedure").  Samples are first lysed and homogenized. The lysate is passed through a gDNA Eliminator spin column, ethanol is added to the flow-through, and the sample is applied to an RNeasy MinElute spin column. RNA binds to the membrane and contaminants are washed away. High quality RNA is eluted in 30 µl, or more, of water.

Different protocols are available for different starting materials. The protocols differ mainly in the lysis and homogenization of the sample. Once the sample is applied to the gDNA Eliminator spin column, the protocols are similar. The procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. The RNeasy Plus procedure can be modified to allow the purification of total RNA containing small RNAs, such as miRNA, from cultured cells. 

When disrupting and homogenizing tissues in Buffer RLT Plus (supplied with the RNeasy Plus Mini Kit), excessive foaming may occur. This foaming is substantially reduced by adding Reagent DX (supplied separately) to Buffer RLT Plus before starting disruption and homogenization. Reagent DX has been carefully tested with the kit, and has no effect on RNA purity or on downstream applications.

Applications

RNA purified using the RNeasy Plus Mini Kit is ideally suited for downstream applications that are sensitive to low amounts of DNA contamination, such as quantitative real-time RT-PCR. The purified RNA can also be used in other applications.

Feature
Specifications
Applications Northern, dot, and slot blotting, end-point RT-PCR, quantitative, real-time RT-PCR, array analysis, next-generation sequencing
Elution volume 30-50 µl
Format Spin column
Integrated removal of genomic DNA Yes
Main sample type Tissue, cells
Processing Manual (centrifugation)
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein Total RNA
Sample amount 30 mg (<700 µl)
Technology Silica technology
Time per run or per prep 25 minutes
Yield Varies

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FAQs
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Kit Handbooks
2
gDNA Eliminatorカラムを用いた動物細胞および溶解しやすい動物組織からのトータルRNA精製
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For purification of total RNA from animal cells and easy-to-lyse animal tissues using gDNA Eliminator columns
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Supplementary Protocols
2

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There are two protocols: follow Protocol 1 if you want to purify total RNA containing miRNA, or follow Protocol 2 if you want to purify small RNA (includes miRNA, 5S rRNA, and tRNA) and larger RNA (>200 nt) separately.
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References
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