RNeasy Plus Kits for RNA Isolation

• Efficient gDNA removal with unique gDNA Eliminator columns or plates (no need for DNase)
• High-quality total RNA in minutes using fast and simple extraction protocols
• Phenol-free RNA isolation
• High-throughput processing in 96-well format
• Ideal for sensitive applications such as real-time RT-PCR and RNA-seq

RNeasy Plus Kits are next generation QIAGEN RNA extraction kits, combining high-quality RNA purification with effective gDNA removal with unique gDNA Eliminator columns or plates. Total RNA of high purity and yield is obtained from a wide range of cultured cells and easy-to-lyse tissues. The RNeasy Plus Micro Kit is specially designed for limited amounts of samples and isolates up to 45 µg pure total RNA The RNeasy Plus Mini Kit purifies up to 100 µg total RNA (>200 nt) from a single extraction with efficient gDNA removal. Both kits can be automated on the QIAcube Connect and samples can be conveniently stabilized using RNAprotect Tissue Reagent or Allprotect Tissue Reagent and efficiently disrupted using a TissueRuptor II or TissueLyser II or LT system (available separately). The RNeasy Plus 96 Kit is for high-throughput purification of total RNA from cultured cells grown in 96-well plates. 

RNA extraction from cells and tissues with RNeasy Plus Micro and Mini Kits 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.

RNeasy Plus Micro and Mini Kits also provide an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. For isolation of 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.
The RNeasy Plus 96 Kit is compatible with a wide range of cell lines, providing purification of high-quality RNA with Agilent RIN values of close to 10 (see figure " High-quality RNA"). Complete genomic DNA removal by the kit (see figure "Effective genomic DNA removal") enables specific transcript detection in highly sensitive applications, such as quantitative, real-time RT-PCR analysis of low-abundance targets. In addition, reliable RNA purification from 10² to 10⁶ cells allows real-time RT-PCR quantification over a wide dynamic range (see figure " Reliable RNA purification").

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 QIAwave RNA Plus Mini Kit is an eco-friendlier version of our standard RNeasy Plus Mini Kit. Reducing plastic and cardboard usage by up to 47% and 42%, respectively, this version incorporates waste tubes made from 100% post-consumer recycled plastic that can be reused during the procedure. The QIAwave buffers are provided as concentrates, decreasing plastic use by up to 90% per bottle. Despite the visual differences, the QIAwave Kit remains user-friendly, with chemistry and performance identical to the standard kit's.

In partnership with My Green Lab, we've also assessed the environmental impact of the RNeasy Plus Mini Kit (50/250) and the QIAwave RNA Plus Mini Kit (50/250). My Green Lab ACT (accountability, consistency, and transparency) environmental impact factor labels are designed to evaluate and score products on several sustainability criteria. These include: 

• Manufacturing
• Responsible chemical management 
• Sustainable content within products and packaging materials 
• Disposal of the packaging at the end of life

Products are scored from 1 to 10 except for energy and water consumption, which are scored as 1 point per kWh or gallon, respectively. A low score means a lower environmental impact (see figures "RNA Plus Mini Kits ACT environmental impact factor label  US (  50/  250),  EU (  50/  250) and UK (  50/  250)."

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 A 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.

RNeasy Plus Mini standard protocols can also be executed using the TRACKMAN Connected system, paired with PIPETMAN M Connected pipettes, both from Gilson. The TRACKMAN Connected system guides researchers through the RNeasy Plus Mini protocols while automatically adjusting the Bluetooth-enabled PIPETMAN M Connected pipette settings. Each step of the protocol execution is recorded to accelerate reporting by generating a comprehensive run report. Download more information.

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

The RNeasy Plus Micro Kit is suitable for small samples, including:

• Laser-microdissected cryosections
• Fine-needle aspirates

The ease with which RNA is purified in 96-well format makes the RNeasy Plus 96 Kit well suited for high-throughput gene expression analysis in areas such as:

• Drug discovery
• Biomedical research