Isolate nuclear and cytoplasmic RNA and protein from cultured cells
No phenol extraction or alcohol precipitation
Ambion's new PARIS Kit uses a fast, simple procedure based on Ambion's RNAqueous technology to isolate 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 resulting protein and RNA samples are suitable for various downstream applications, reducing time, cost, and variability between independent experimental samples.
Protein And RNA Isolation System (PARIS)
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, e.g. performing further in vitro functional assays, analyzing a regulatory pathway, or simply correlating RNA and protein expression levels. 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 Ambion scientists have developed a unique Protein And RNA Isolation System (PARIS) that allows researchers to isolate both RNA and protein from the same experimental sample (Figure 1).
Figure 1. Schematic of PARIS Procedure.
Using PARIS, RNA and protein can be isolated simultaneously from whole cell lysates. Alternatively, RNA and protein can be isolated from separate nuclear and cytoplasmic fractions (see "Application: Nuclear vs. Cytoplasmic Fractionation" at right). 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. This solution contains a high concentration of guanidinium thiocyanate, a strong chaotropic denaturant that rapidly inactivates cellular ribonucleases. 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, especially if using primers that do not flank introns, or for genes that have processed pseudogenes. Ambion's DNA-free DNase Treatment and Removal Reagents (available separately) 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.
Each protein fraction can also be used directly for most common applications, including functional assays, immunoprecipitation, Western blotting or two-dimensional gel electrophoresis (Figure 2 below). For experiments such as gel shift assays or enzymatic assays, protein samples are usually concentrated enough to be diluted in the final reaction buffer. The Cell Disruption Buffer and Cell Fractionation Buffer have been specially designed to be compatible with downstream analyses.
The PARIS Kit includes sufficient reagent to perform 50 purifications. The kit also comes with a comprehensive Instruction Manual and RNase-free microfuge tubes, glass fiber filters and reagents for RNA isolation. Each purification can accommodate 1 to 75 mg of tissue or 100 to 107 cells.PARIS is Compatible with RNAlater and RNAlater-ICE Treated Tissue.
Figure 2. 2D Analysis of Proteins Isolated Using the PARIS Kit. Protein samples were resolved using a pH 4-7 IPG gel followed by 8-16% SDS-PAGE. Top: Total protein (~25 g) from HeLa cells stained with SYPRO Ruby. Bottom: Total protein (~125 g) from mouse brain stained with Coomassie Blue.
Figure 3. RNA and Protein Isolation from Stabilized Samples. (A) Isolation from ~30 mg of mouse liver, brain or kidney 5 days after collection. Samples were either kept frozen at -80C, stored in RNAlater at 4C, or snap frozen and then stored in RNAlater-ICE at -20C. (B) Isolation from 106 HeLa cells freshly harvested, kept 2 days in RNAlater at RT, or in RNAlater-ICE at -20C.
Figure 4. Analysis of the RNAi Effect. (A) 5 x 104 HeLa cells were transfected in a 24 well plate with the indicated plasmids. 48 hours after transfection, total RNA and protein were isolated with the PARIS Kit and analyzed by denaturing agarose gel electrophoresis, Northern blot, or Western blot. SCR represents a scrambled siRNA sequence. (B) 5 x 105 Schneider's Drosophila cells were treated for 48 hours in a 6 well plate with 10 nM of the indicated dsRNA prepared with Ambion's MEGAscript RNAi Kit. Total RNA and protein were isolated with the PARIS Kit and analyzed by Northern blot and Western blot.
Figure 5. Efficient Cellular Fractionation. (A) RNA and protein were isolated from 106 HeLa cells using the PARIS Kit. To allow direct comparison between RNA isolated from different cellular compartments, the same volume of each fraction (~5%) was analyzed by denaturing agarose gel electrophoresis, Northern blot, or Western blot. (B) 3 x 105 HeLa cells were transfected in duplicate in 6 well plates with 1 g of luciferase mRNA and the relative luciferase activity in each protein fraction was measure 18 hours after transfection. Luciferase mRNA was prepared with Ambion's mMESSAGE mMACHINE and Poly(A) Tailing Kits.