RNeasy Plus Micro and Mini Kits
For phenol-free total RNA extraction from cells/tissues with removal of genomic DNA contamination
The RNeasy Plus Kits are next generation QIAGEN RNA extraction kits, combining high-quality RNA purification with effective on-column gDNA removal. Total RNA of high purity and yield is obtained from a wide range of cultured cells and easy-to-lyse tissues. The RNeasy Plus Mini Kit purifies up to 100 µg total RNA (>200 nt) from a single extraction with efficient gDNA removal. The RNeasy Plus Micro Kit is specially designed for limited amounts of samples and isolates up to 45 µg pure total RNA. The kits can be automated on the QIAcube and samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent (available separately).
RNA extraction from cells and tissues with the RNeasy Plus procedure allows high, reproducible RNA yields and efficient genomic DNA elimination 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 tissues and cultured cells.
These RNA isolation kits also provide an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. For isolating total RNA containing small RNAs (e.g., miRNA) from cultured cells, additional RNA isolation protocols are available and described in the RNeasy Plus Micro Handbook and as a separate Supplementary Protocol to the RNeasy Plus Mini Kit.
Cells and easy-to-lyse tissues are first 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 that, 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 spin column. These specialized columns contain a silica membrane that specifically binds RNA from lysed cells.
For fatty and difficult-to-lyse tissues, the RNeasy Plus Universal Kits are recommended. For high-throughput total RNA purification, the RNeasy Plus 96 Kit is recommended. For isolation of total RNA including miRNA from other sample types, we recommend miRNeasy Kits.
The RNeasy Plus Micro Kit purifies total RNA from up to 5 x 105 cells or 5 mg tissue, and the RNeasy Plus Mini Kit isolates total RNA from up to107 cells or 30 mg tissue. A short workflow enables RNA isolation 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 spin column. RNA binds to the membrane and contaminants are washed away. High-quality RNA is eluted in as little as 14 µl water using the RNeasy Plus Micro Kit or 30 µl water using the RNeasy Plus Mini Kit.
Different RNA extraction protocols are available for different starting materials. They 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.
When disrupting and homogenizing tissues in Buffer RLT Plus (supplied with the RNeasy Plus Kits), 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 kits, and has no effect on RNA purity or on downstream applications.
RNA purified using the RNeasy Plus Kits is ideally suited for downstream applications that are sensitive to low amounts of DNA contamination, such as quantitative real-time RT-PCR and RNA-Seq. The purified RNA can also be used in other applications.
The RNeasy Plus Micro Kit is suitable for small samples, including:
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