Description
The RNeasy Plus Universal Mini Kit integrates fast, convenient RNA purification with effective elimination of genomic DNA. Optimized protocols enable purification of high-quality RNA from any type of tissue, even difficult-to-lyse tissues. The kit includes QIAzol Lysis Reagent for lysing fatty tissue and other types of tissue and RNeasy spin columns for purifying high-quality RNA. The RNeasy Plus Universal Mini Kit can be automated on the QIAcube Connect. The RNeasy 96 Universal Tissue Kit enables total RNA purification in 96-well format from any tissue. Tissues are efficiently lysed and homogenized in QIAzol Lysis Reagent, and high-quality RNA is purified using silica-membrane RNeasy 96 plates. Manual purification is carried out using the QIAGEN Centrifuge 4-16KS and, optionally, the QIAvac 96 vacuum manifold. Automated purification using the related RNeasy 96 Universal Tissue 8000 Kit is performed on the BioRobot Universal System. Tissue samples can be conveniently stabilized using Allprotect Tissue Reagent, and efficiently disrupted using the TissueLyser II. Nonfatty tissues can be stabilized using the RNAprotect Tissue Reagent.
The RNeasy Plus Universal procedure allows high, reproducible RNA yields from any type of tissue, including fatty tissues and difficult-to-lyse tissue (see table “Typical yields of total RNA using the RNeasy Plus Universal Mini Kit”). Yields can vary due to factors such as species and developmental stage, especially with adipose tissues. Typical yields of total RNA with RNeasy Plus Universal Kits Mouse/rat tissue (10 mg) Yield of total RNA ((µg) Adipose tissue 0.5–2.5 Brain 5–20 Heart 5–25 Intestine 10–60 Kidney 5–40 Liver 15–80 Lung 5–15 Muscle 5–35 Skin 2–5 Spleen 15–100 Analysis of purified RNA by real-time RT-PCR, and on the QIAxcel system, demonstrates high-quality RNA with good integrity (see figure "High-quality RNA"). gDNA Eliminator Solution and RNeasy technologies efficiently remove most of the genomic DNA contamination without DNase treatment (see figure "Effective removal of genomic DNA"). Compared with standard silica-membrane procedures, the RNeasy 96 system provides higher yields of total RNA for all tissue types (see table “Typical total RNA yields using the RNeasy 96 Universal Tissue Kit”). Typical total RNA yields using the RNeasy 96 Universal Tissue Kit Tissue RNA yield* (µg per 10 mg of tissue) Kidney 5–40 Liver 15–80 Lung 5–15 Heart 5–25 Muscle 5–35 Brain 5–20 Adipose tissue 0.5–2.5 Spleen 15–100 Intestine 10–60 Skin 2–5 High-quality RNA can be isolated from tissues preserved either by freezing or by stabilization with RNAprotect at room temperature (see figure "High-quality total RNA from rat liver and muscle tissue"). Intact RNA purified from difficult-to-lyse fibrous and fatty tissues is suitable for downstream applications (see figures "High-quality RNA from skin / adipose / liver / intestine tissue "and "Real-time analysis of high-quality RNA from rat brain").
The RNeasy Plus Universal Mini procedure enables purification of RNA from any type of tissue, including fatty or difficult-to-lyse tissues. The RNeasy Plus Universal Mini Kit integrates phenol/guanidine-based sample lysis and silica-membrane purification of total RNA. QIAzol Lysis Reagent, included in the kits, is a monophasic solution of phenol and guanidine thiocyanate, designed to facilitate lysis of all kinds of tissues and to inhibit RNases. The highly efficient lysis and the subsequent removal of contaminants by organic phase extraction enable the use of larger amounts of tissue with RNeasy spin columns. gDNA Eliminator solution effectively removes genomic DNA contamination and significantly improves separation of DNA into the interphase. The RNeasy 96 Universal Tissue procedure enables high-throughput purification of RNA from any animal or human tissue sample. Compared with standard silica-membrane procedures, the RNeasy 96 Universal Tissue Kit provides higher yields of total RNA for all tissue types. The RNeasy procedure enriches for RNA >200 bases long, providing a yield of total RNA that does not include 5.8S rRNA, tRNA, and other low-molecular weight RNAs, which make up 15–20% of total cellular RNA.
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.
