Principle and procedure
A short workflow enables RNA purification with genomic DNA removal in less than 25 minutes (see flowchart "RNeasy Plus procedure"). Total RNA is purified from up to 107 cells or 30 mg tissue. Cells and easy-to-lyse tissues are lysed in Buffer RLT Plus, which provides optimal conditions for sample lysis and binding of genomic DNA to gDNA Eliminator spin columns. This allows high, reproducible RNA yields and effective elimination of genomic DNA contamination for sensitive applications (see figures "Effective elimination of genomic DNA contamination", "High, reproducible RNA yields", and "Effective genomic DNA removal"). After genomic DNA removal, high-quality RNA is purified using RNeasy spin columns. Total RNA with Agilent RIN values of close to 10 is routinely obtained from cultured cells.
RNA purified using the RNeasy Plus Mini Kit is of high quality and ready to use in any downstream application, such as real-time RT-PCR and array analysis (see figure "Array-ready RNA").
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 at a final concentration of 0.5% (v/v) 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 such as real-time RT-PCR. Buffer RLT Plus containing Reagent DX can be stored at room temperature (15–25ºC) for at least 9 months.