The RNeasy Plus procedure integrates QIAGEN’s innovative technology for selective binding of double-stranded DNA with well-established RNeasy technology. Efficient purification of high-quality RNA is guaranteed, without the need for additional DNase digestion. The purified RNA is ready to use and is ideally suited for downstream applications that are sensitive to low amounts of DNA contamination, such as quantitative, real-time RT-PCR.
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 or gDNA Eliminator 96 plate 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 or 96-well plate. These specialized columns and 96-well plates contain a silica membrane that specifically binds RNA from lysed cells.
For fatty and difficult-to-lyse tissues, RNeasy Plus Universal Kits are 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 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.
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.
High-throughput RNA purification is achieved through the use of RNeasy 96 plates. Prior to application to the RNeasy 96 plate, cell lysates are first passed through a gDNA Eliminator 96 plate, (see figure " RNeasy Plus 96 procedure"). The gDNA Eliminator 96 plates are conveniently processed using a centrifuge (Centrifuge 4-16). RNA purification using RNeasy 96 plates is manual, and comprises 3 simple steps: bind, wash, and elute. The plates are rapidly and conveniently processed using either a centrifuge (Centrifuge 4-16 and Plate Rotor 2 x 96) or a combination of vacuum (QIAvac 96) and centrifuge. Up to 2 x 106 cells can be used as starting material when processing plates using a centrifuge, while up to 1 x 106 cells can be used when processing plates using a vacuum and centrifuge. 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.