- Isolate small RNA-containing total RNA and native protein from the same sample
- Use 100-107 cultured cells or up to 100 mg of many mammalian tissues
- Enrich for small RNA <200 nt to increase sensitivity in downstream analyses
- Simple 30 minute procedure
- Ideal for correlating mRNA, miRNA or siRNA, and protein levels
Two Kits in One: Isolates Small RNA and Protein
The mirVana PARIS Kit provides a unique and versatile procedure that permits quantitative recovery of native protein and all RNA species, including small RNAs such as miRNA, siRNA, snRNA, and snoRNA, from the same sample. The kit also includes a procedure to enrich the population of small RNAs <200 nt, which can dramatically enhance sensitivity in downstream applications such as Northern analysis or solution hybridization with the mirVana™ miRNA Detection Kit. The ability to isolate small RNA, total RNA, and protein from the same sample greatly simplifies the correlation of protein levels with mRNA and miRNA or siRNA levels.
How the Kit Works
The mirVana PARIS Kit procedure begins with homogenization of samples with a special Cell Disruption Buffer that includes nonionic detergent (see schematic, Figure 2). Protein remains intact, so a portion of the lysate can be used directly for common applications such as Western blotting, two-dimensional gel electrophoresis, or enzymatic assay. The remainder of the lysate is used for RNA isolation with a procedure that combines the advantages of organic and solid-phase extraction, while avoiding the disadvantages of both. First, total RNA is extracted using a highly efficient acid-phenol:chloroform step. Next, the RNA is purified over a glass-fiber filter (GFF) using conditions that recover small-RNA containing total RNA, or that separate small RNA species less than 200 nt in length from longer RNA species such as mRNA. Reagents and detailed instructions for both sets of conditions are supplied. Because the RNA is eluted in a small volume, no alcohol precipitation step is required. High yields of ultra-pure RNA can be prepared in about 30 min. The high quality RNA recovered can be used in any application, including RT-PCR, blot hybridization, solution hybridization assays (e.g., the mirVana miRNA Detection Kit) or RNA amplification and microarray analysis.
Compatible with Cultured Cells and Many Tissues
The mirVana PARIS Kit was specifically designed to quickly process cultured cell samples. However, the kit can also be used to isolate RNA and protein from many animal tissues, and is compatible with Ambion's RNAlater® Tissue Collection: RNA Stabilization Solution. An example of the kit's utility with mouse brain and kidney tissues is depicted in Figure 3. Please note that the mirVana PARIS Kit is not recommended for use with tissues that contain very high levels of ribonucleases, such as pancreas or spleen. (The mirVana miRNA Isolation Kit is a better choice for these tissue types, although that kit is not designed to recover protein and small RNA from the same sample. See Figure 4 for a kit comparison chart.)
Purify siRNA-Containing Total RNA and Protein for RNAi Applications
The mirVana PARIS Kit is ideal for preparing samples for analysis after induction of RNA interference (RNAi). Researchers often need to assess knockdown of both a target mRNA and its corresponding protein. Ascertaining siRNA levels within cells after siRNA transfection or expression can also be important. Figure 5 demonstrates the effectiveness of the mirVana PARIS Kit for preparing samples for this type of experiment. Because the siRNA, target mRNA, and target protein levels can all be assessed from the same sample, fewer samples are needed for the experiment. Furthermore, concern about sample-to-sample variation is eliminated.
Kit Contents
The mirVana PARIS Kit includes reagents for 40 isolations of protein and small RNA-containing total RNA or 20 isolations of protein and fractions enriched and depleted in small RNA species.
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