The Ambion® PARIS™ system is for the isolation of both RNA and native protein from the same experimental sample. The kit also permits separation of nuclear and cytoplasmic fractions prior to RNA and/or protein isolation. The kit contains sufficient reagents for 50 purifications from1 to 75 mg of tissue or 100 to 107 cells each.
• Fast, simple procedure
• Isolate nuclear and cytoplasmic RNA and protein from cultured cells
• Isolate total RNA and protein from cultured cells or tissues
• No phenol extraction or alcohol precipitation
• Reduces time, cost, and variability between independent experimental samples
Isolation of high-quality RNA is the first step for a variety of gene expression analyses. Very often, complementary studies at the protein level are also required. Usually these analyses are performed using different aliquots of the same experimental sample. However, when working with rare, difficult to obtain, or very small samples, it is sometimes impractical to isolate RNA and native proteins independently. In studies involving large numbers of samples, expensive reagents, or inherent variability (e.g., cell transfection), the addition of independent experimental samples is not only costly and time consuming, but may also lead to inconsistent results. To solve these issues, the Protein And RNA Isolation System (PARIS™ system) has been developed. It allows for the isolation of both RNA and protein from the same experimental sample (see Figure). Using the PARIS™ system, RNA and protein can be isolated simultaneously from whole cell lysates. Alternatively, RNA and protein can be isolated from separate nuclear and cytoplasmic fractions. Tissue or cultured cells are first homogenized in ice-cold Cell Disruption Buffer to prepare a total cell lysate. Since the homogenization is performed quickly on ice and in the presence of detergent, both protein and RNA can be purified directly from this lysate. For RNA isolation, a part of the total cell lysate is immediately mixed with an equal volume of Lysis/Binding Solution. Total RNA is then purified from the mixture using an RNA binding glass fiber filter. After three rapid washing steps, high-quality RNA is eluted in a concentrated form. The entire procedure can be completed in less than 20 minutes. Note: This kit is not recommended for tissues with high levels of ribonucleases, such as pancreas.
Compatible with Most Downstream Applications
The RNA isolated from total, nuclear, or cytoplasmic fractions with the PARIS™ procedure can be used in a variety of downstream applications, including blot hybridization, in vitro translation, cDNA synthesis, and RT-PCR. A DNase I treatment is recommended for RNA that will be used for RT-PCR experiments (see Accessory Products). Protein fractions can be used directly for most common applications, including functional assays, immunoprecipitation, western blotting, or two-dimensional gel electrophoresis (see Figures).
TURBO™ DNA-free™ or DNA-free™ DNase Treatment and Removal Reagents (SKU #AM1907 and AM1906, respectively) are ideal to quickly remove trace amounts of DNA from the total and nuclear RNA samples without phenol extraction or alcohol precipitation. The cytoplasmic RNA fraction is virtually free of DNA contamination.