RNeasy Plus Mini Kit
For purification of up to 100 µg total RNA from cells/tissues using gDNA Eliminator columns
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.
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.
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.
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.
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.
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