miRNeasy Tissue/Cells Advanced Micro

QIAGEN offers a phenol-free solution for purification of total RNA, including miRNA and other small RNA, from cultured cells and various animal and human tissues. The workflow combines guanidine-based sample lysis with an inhibitor removal step and silica-membrane-based purification. Buffer RLT, included in the kit, contains guanidine thiocyanate to facilitate lysis and denature RNases and other proteins, protecting RNA in lysed samples from degradation.

After a thorough disruption and homogenization step, Buffer AL is added to the lysate to optimize gDNA removal by the gDNA Eliminator Spin Column. Buffer RPP is then added to the flow-through to precipitate inhibitors (mostly proteins that are highly concentrated in tissue and cell samples) by centrifugation. Isopropanol is added to provide binding conditions for RNA molecules from approximately 18 nucleotides upwards, and the sample is applied to an RNeasy UCP MinElute Spin Column, where total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA, including miRNA and other small RNA, is eluted in a small volume of RNase-Free Water.

The kit enables efficient enrichment of RNA down to approximately 18 nucleotides in size and efficiently purifies RNA from up to 5 mg frozen tissue or 1 x 10^6 cells, while also working with significantly lower amounts of starting material. Total RNA with Agilent RIN or QIAxcel Connect System RIS values close to 10 is routinely obtained from tissues and cultured cells. The default elution volume is 20 µl, with a minimum of 10 µl. Typical applications include NGS, real-time RT-PCR, microarray analysis, RNA-seq, and Northern blot analysis. Purification can be fully automated on QIAcube Connect or the classic QIAcube, and the kit is compatible with the miRCURY LNA miRNA PCR systems for sensitive and specific quantification of miRNA expression. The kit is shipped at ambient temperature; the RNeasy UCP MinElute spin columns should be stored at 2–8°C and all other components can be stored dry at room temperature, remaining stable for at least 9 months. The product is intended for molecular biology applications and is not intended for the diagnosis, prevention, or treatment of a disease.