Navigation Links
A Better Way to Isolate RNA AND Protein From the Same Sample


Isolate total RNA and protein from cultured cells or tissues

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.
Many researchers use the RNA stabilization reagent RNAlater to protect RNA integrity immediately after cell or tissue collection, to stabilize RNA during long period of storage, or to circumvent the need to grind frozen tissue into a powder prior to RNA isolation. (See the article "RNArticles: Keeping RNA Intact Prior to RNA Isolation - RNAlater" for some of its many applications.) Ambion's new RNAlater-ICE permits transition of previously frozen samples to a state compatible with handling at room temperature and standard homogenization methods (see the article"Stop Grinding Frozen Tissue" in this issue). As shown in Figure 3, the PARIS procedure is compatible with both of these tissue collection/RNA stabilization reagents for both cells and tissue. After homogenization, protein samples are readily available for applications that do not require native functional protein, such as Western blot analysis.

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.


back to top


Ordering Information Cat# Product Name Size 1921 PARIS Kit 50 purifications
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Better RNA Amplification
2. Designing a Better siRNA
3. RNA Amplification Just Got Better: MessageAmp II
4. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
5. Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit
6. Isolate It All: siRNA miRNA Total RNA Native Protein
7. Isolate Total RNA and Protein From the Same Sample
8. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
9. A New C-Terminal GST Vector for Protein Production in S. pombe
10. New Mammalian Two-Hybrid System Detects Protein-Protein Interactions
11. Antibodies for Studying NMDA Receptor Protein Expression and Synapse-Specific Immunolabeling
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Better Way Isolate RNA AND Protein From the Same Sample

(Date:7/30/2015)... ... , ... The 2015 Market Research Report on the Global Propanol Industry ... Propanol market with a focus on the Chinese situation. Major companies included in the ... Chemical, Sanofi, Royal Dutch Shell, LG Chem and Zhejiang Xinhua Chemical. , The report ...
(Date:7/30/2015)... July 30, 2015   Senomyx , Inc. (NASDAQ: ... science technologies to discover, develop, and commercialize novel flavor ... today reported financial results for the second quarter 2015. ... 2015, we remain on track to achieve our commercial ... Executive Officer of the Company. "Since our last quarterly ...
(Date:7/30/2015)... July 30, 2015  Discovery Laboratories, Inc. (Nasdaq: ... received the second $1.0M tranche under a previously ... grant valued at up to $3.0 million to ... surfactant as a potential medical countermeasure to mitigate ... was awarded an initial $1.0 million under this ...
(Date:7/29/2015)... July 30, 2015 Sanofi, ... publie ses résultats pour le premier semestre 2015. ... les résultats. Visionner l,interview vidéo et ... Au sommaire de l,interview :  ... Moteurs de croissance - Diabète ...
Breaking Biology Technology:Global Propanol Market Technical Data and Manufacturing Analysis 2020 2Global Propanol Market Technical Data and Manufacturing Analysis 2020 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 2SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 3SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 4SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 5SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 6SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 7SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 8SENOMYX ANNOUNCES SECOND QUARTER 2015 FINANCIAL RESULTS 9Discovery Labs Receives $1.0 million under an SBIR Grant in Continuing Support of the Development of Aerosolized KL4 Surfactant to Address Radiation-Induced Lung Injury 2Discovery Labs Receives $1.0 million under an SBIR Grant in Continuing Support of the Development of Aerosolized KL4 Surfactant to Address Radiation-Induced Lung Injury 3
... 7, 2011 Reportlinker.com announces that a new ... Industrial Biotechnology China News 1103 ... The Chinese central government was reported to continue ... the Twelfth Five-Year (2011-2015) Plan. This act is ...
... and industrial research scientists and engineers, as well ... encounter a steep learning curve when developing common ... book just published by Springer promises to be ... The new MEMS Materials and Processes Handbook is ...
... The U.S. Food and Drug Administration today approved ... tool to treat brain aneurysms without performing open surgery. ... An aneurysm is a weak, enlarged area in an ... blood pressure can cause it to rupture, which can ...
Cached Biology Technology:Reportlinker Adds Industrial Biotechnology China News 1103 2Reportlinker Adds Industrial Biotechnology China News 1103 3MEMS Materials and Processes Handbook -- a comprehensive, practical resource 2FDA Approves New Device to Treat Brain Aneurysms 2FDA Approves New Device to Treat Brain Aneurysms 3
(Date:7/13/2015)... OXFORD, Conn. , Jul. 13, 2015 /PRNewswire/ ... the "Company"), a biometric authentication company focused on ... it has filed provisional patent 62/188684 for ... NXT-ID achieves another new convenient and secure method ... expands new, innovative payment methods, introduced with its ...
(Date:7/9/2015)... PLEASANTON, Calif. , July 9, 2015 /PRNewswire/ ... tools, today announcing its acquisition of Avid Nano. ... dynamic light scattering (DLS) systems.    ... world,s smallest, fastest and easiest to use protein ... system that measures a protein,s hydrodynamic size, size ...
(Date:7/8/2015)... , N.J. and NEW YORK , ... and Guidepoint today announced BD & ... healthcare companies with free access to Guidepoint,s expert network ... start-ups that are developing cutting-edge technologies to improve healthcare ... dedicated Guidepoint research manager, each start-up entrepreneur will be ...
Breaking Biology News(10 mins):NXT-ID Patents Behavior-Directed Payments 2NXT-ID Patents Behavior-Directed Payments 3NXT-ID Patents Behavior-Directed Payments 4Unchained Labs just can't get enough, acquires Avid Nano, rocks out the new pUNk! 2BD and Guidepoint Team to Connect Healthcare Start-up Companies With Business and Scientific Experts 2BD and Guidepoint Team to Connect Healthcare Start-up Companies With Business and Scientific Experts 3
... The first State of the Carbon Cycle Report for North ... Science Program, finds the continents carbon budget increasingly overwhelmed by ... tons of carbon into the atmosphere each year, mostly as ... may remove up to half this amount, but these current ...
... In the December 1st issue of G&D, Dr. Hailing ... of a new class of small RNAs in Arabidopsis. ... dubbed long short interfering RNAs (lsiRNAs), and are induced ... liRNAs share some hallmark features with other, previously identified ...
... Researchers from Kaiser Permanente and the California Department of ... nationwide, today will begin enrolling families in the largest ... that may cause autism and other developmental disabilities. ... Early Development, will involve 2,700 children and their parents ...
Cached Biology News:First-ever 'State of the Carbon Cycle Report' finds troubling imbalance 2Largest study to investigate risk factors of autism to begin enrolling families 2
... silencing experiments often call for critical, ... Transfection Agent refines siRNA transfection protocols ... lipid-based formulation can be used to ... are subcultured without increased cytotoxicity. ...
... The CHEMICON® CaspaTag™ Pan-Caspase In Situ ... detection of active caspases in cells undergoing ... only. Not for use in diagnostic or ... Assay Kits use a novel approach to ...
... INTERFERin is a new-generation siRNA transfection ... efficiency at 1 nM siRNA in a ... with excellent cell viability. Using low siRNA ... effects observed when transfecting siRNA at higher ...
... Cell and Tissue Staining Kits are intended ... range of histological and cytological specimens. These ... antibodies manufactured by either R&D Systems or ... on the formation of the Avidin-Biotin Complex ...
Biology Products: