miRNeasy Tissue/Cells Advanced Kits – microRNA Isolation

• Phenol-free protocol – no need for phase separation or working under a hood
• Fast and convenient procedure due to the advanced chemistry and gDNA Eliminator Columns
• Efficient enrichment of miRNA and RNAs <200 nucleotides
• Consistent, pure RNA for all downstream applications

The miRNeasy Tissue/Cells Advanced Kits are the second generation of the well-established miRNeasy Kits. These improved versions combine a phenol-free advanced chemistry with gDNA Eliminator Columns or gDNA 96 Eliminator Plates for safe and convenient RNA isolation. They enable purification of total RNA, including small RNAs ≥18 nucleotides, from animal tissues and cells, including small samples. The kits use spin columns for low-throughput RNA purification. The miRNeasy Tissue/Cells Advanced Mini Kit can be automated on the QIAcube Connect.

The miRNeasy Tissue/Cells Advanced Kits are phenol-free alternatives to classical miRNeasy Kits without compromising on RNA yields and quality (see figures "High recovery of miRNA without the need for phenol", " High recovery of miR16 from tissues and cells without the need for phenol using miRNeasy Tissue/Cells Advanced Micro", and " High recovery of miRNA without phenol using the miRNeasy 96 Tissue/Cell Advanced Kit"). The miRNeasy Tissue/Cells Advanced Kits enable efficient enrichment of RNA down to approximately 18 nucleotides in size. 

The miRNeasy Tissue/Cells Advanced Micro Kit efficiently purifies RNA from up to 5 mg frozen tissue or 1 x 10⁶ cells but also works with significantly lower amounts of starting material. The miRNeasy Tissue/Cells Advanced Mini Kit efficiently purifies RNA from up to 30 mg frozen tissue or 1 x 10⁷ cells. Up to 50 mg tissue and 1 x 10⁷ cells can be processed with the 96-well miRNeasy 96 Tissue/Cell Advanced Kit.

The miRNeasy Tissue/Cells Advanced Kits include the gDNA Eliminator procedure allowing high, reproducible RNA yields and efficient genomic DNA elimination for sensitive applications (see figures " The miRNeasy Tissue/Cells Advanced Mini Kit shows higher performance," " Total RNA extraction: effective genomic DNA removal", and "gDNA 96 Eliminator plates ensure efficient genomic DNA removal"). Total RNA with Agilent RIN or QIAxcel Connect System RIS values close to 10 is routinely obtained from tissues and cultured cells. Unlike kits from other suppliers, the miRNeasy Tissue/Cells Advanced Kits combine ease-of-use and a phenol-free protocol without any compromise on RNA quality or yield (See figures " The miRNeasy Tissue/Cells Advanced Mini Kit shows better performance compared to kits from other supplies," " Competitive performance," " Fast procedure," and The miRNeasy 96 Tissue/Cell Advanced Kit offers excellent performance").

The miRNeasy Tissue/Cells Advanced Kits combine a guanidine-based sample lysis with an inhibitor removal step and a silica-membrane-based purification of total RNA, which includes miRNA and other small RNA. Buffer RLT, included in the kit, contains guanidine thiocyanate which facilitates lysis and denaturation of RNases and other proteins. Therefore, RNA in samples lysed in buffer RLT are stable and protected from degradation.

Sample lysis is achieved by adding Buffer RLT to tissue or cell samples followed by a thorough disruption and homogenization step to ensure complete lysis and inactivation of RNases (see figure " miRNeasy Tissue/Cells Advanced Kit procedure"). Next, Buffer AL is added to the lysate to optimize gDNA removal by the gDNA Eliminator Spin Column or gDNA 96 Eliminator Plate. Buffer RPP is added to the flow-through to precipitate inhibitors (mostly proteins that are highly concentrated in tissue and cell samples and might interfere with the RNA isolation or the downstream analysis) by centrifugation. In case of the miRNeasy 96 Tissue/Cell Advanced Kit, a Proteinase K step is included to remove proteins. The supernatant containing the total RNA is transferred into a new reaction tube, and isopropanol is added to provide appropriate binding conditions for all RNA molecules ≥18 nucleotides. The sample is then applied to an RNeasy Mini Spin Column (miRNeasy Tissue/Cells Advanced Mini), an RNeasy UCP MinElute Spin Column (miRNeasy Tissue/Cells Advanced Micro), or to an RNeasy® 96 Plate (miRNeasy 96 Tissue/Cell Advanced), where the total RNA binds to the membrane and all contaminants are efficiently washed away. High-quality RNA, including miRNA and other small RNA, are eluted in a small volume of RNase-Free Water.

The miRNeasy Tissue/Cells Advanced Kits allow purification of miRNA and total RNA for a variety of applications, including:

• RNA-seq
• Quantitative, real-time RT-PCR
• Microarray analysis
• Northern blot analysis