RNeasy Plus 96 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").

Cells are first lysed and homogenized in a highly denaturing guanidine-isothiocyanate–containing buffer, which immediately inactivates RNases to ensure isolation of intact RNA. The lysates are then passed through a gDNA Eliminator 96 plate. This 96-well plate, 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 samples are applied to an RNeasy 96 plate. Each well of these specially designed 96-well plates contains a silica membrane that specifically binds RNA from lysed cells.

High-throughput RNA purification is achieved through the use of RNeasy 96 plates. Prior to application to theRNeasy 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.

The purified total RNA, which represents the transcriptome state of the cell, 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.

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

Drug discovery

Biomedical research