The RNeasy Plus Universal procedure is easy to follow and, compared to using phenol/chloroform to extract total RNA with a subsequent cleanup procedure, much faster and more efficient with better RNA purity and yield (see flowchart " RNeasy Plus Universal Mini procedure"). Total RNA is purified from up to 50 mg tissue using the RNeasy Plus Universal Mini Kit.
Tissue samples are homogenized in QIAzol Lysis Reagent using the TissueRuptor II, TissueLyser LT, or TissueLyser II. After addition of gDNA Eliminator Solution and chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. The upper, aqueous phase with RNA is collected, mixed with ethanol, and RNA is purified using RNeasy spin columns. Total RNA binds to the spin column membrane, and phenol and other contaminants are efficiently washed away. High-quality RNA is eluted in RNase-free water.
The procedure provides an enrichment for mRNA since most RNAs <200 nucleotides (such as rRNAs and tRNAs) are excluded. The RNeasy Plus Universal procedure can be modified to allow the purification of total RNA containing small RNAs, such as miRNA, from cultured cells.
The optimized RNeasy 96 Universal Tissue procedure integrates TissueLyser / QIAzol processing with RNeasy 96 purification in an easy-to-follow protocol (see flowchart " RNeasy 96 Universal Tissue procedure"). Efficient high-throughput disruption using the TissueLyser II provides parallel disruption and homogenization, with lysis of up to 192 samples in QIAzol Lysis Reagent. Protocol steps are performed manually using the QIAGEN 96-Well-Plate Centrifugation System, with optional use of the QIAvac 96 vacuum manifold.