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:10/20/2014)... , A major pan-European survey ... delivery of care has revealed changing trends in many ... the provision of healthcare services across the continent. The ... European Gastroenterology (UEG), have been announced today and led ... the burden of GI disorders across Europe ...
(Date:10/20/2014)... , Oct. 20, 2014 PureTech , ... tackling big healthcare problems, announced today the closing ... with participation from Invesco Perpetual, a $120 Billion ... to drive PureTech,s existing pipeline forward and to ... has the scientific creativity to really go for ...
(Date:10/20/2014)... October 20, 2014 Earle ... LLC, announced today that Ellen Teplitzky, an experienced ... for the pharmaceutical industry, has joined the firm ... its legal services practice. NDA Partners provides ... witness and testimony, to top law firms and ...
(Date:10/19/2014)... The Latin American hardware encryption display market report ... analysis and forecast of revenue. This market was valued ... reach $2,366.8 million by 2018, at a CAGR of ... TOC of the Latin American hardware encryption market report ... It also provides a glimpse of the segmentation of ...
Breaking Biology Technology:Major Survey Reveals Changing Trends and Inequalities in Healthcare Provision for Gastrointestinal Disorders Across Europe 2Major Survey Reveals Changing Trends and Inequalities in Healthcare Provision for Gastrointestinal Disorders Across Europe 3Major Survey Reveals Changing Trends and Inequalities in Healthcare Provision for Gastrointestinal Disorders Across Europe 4Major Survey Reveals Changing Trends and Inequalities in Healthcare Provision for Gastrointestinal Disorders Across Europe 5PureTech Announces Expansion with Oversubscribed $55M Funding Round 2PureTech Announces Expansion with Oversubscribed $55M Funding Round 3PureTech Announces Expansion with Oversubscribed $55M Funding Round 4PureTech Announces Expansion with Oversubscribed $55M Funding Round 5PureTech Announces Expansion with Oversubscribed $55M Funding Round 6NDA Partners Appoints Ellen Teplitzky, JD as Director of its Legal Services Practice 2The Latin American hardware encryption display market is expected to reach $2,366.8 million by 2018 - New Report by MicroMarket Monitor 2The Latin American hardware encryption display market is expected to reach $2,366.8 million by 2018 - New Report by MicroMarket Monitor 3
... authorizations of $570.6 million, a sequential increase of 20.6 ... - Cash flow from operations of $61.6 million, resulting in $146.3 million ... cash flow from operations year-to-date ... of $0.32 within guidance range, WILMINGTON, N.C., Oct. 23 PPD, Inc. ...
... Oct. 23 Valeritas, LLC, a medical,technology company ... delivery solutions, announced today that John Timberlake,Executive Vice ... awarded,the distinguished Executive of Year award by the ... patients with diabetes in New,Jersey. "John,s continued ...
... (OTC Bulletin Board: CSBR), a development stage,biotechnology company, ... of the Head and Neck Cancer Research Division ... as Chairman of the Board,replacing James Martell who ... was appointed to serve on the Company,s Board ...
Cached Biology Technology:PPD Reports Third Quarter 2007 Financial Results; Announces Increase in Annual Cash Dividend From $0.12 to $0.40 Per Year 2PPD Reports Third Quarter 2007 Financial Results; Announces Increase in Annual Cash Dividend From $0.12 to $0.40 Per Year 3PPD Reports Third Quarter 2007 Financial Results; Announces Increase in Annual Cash Dividend From $0.12 to $0.40 Per Year 4PPD Reports Third Quarter 2007 Financial Results; Announces Increase in Annual Cash Dividend From $0.12 to $0.40 Per Year 5PPD Reports Third Quarter 2007 Financial Results; Announces Increase in Annual Cash Dividend From $0.12 to $0.40 Per Year 6John Timberlake, Valeritas' Executive Vice President and General Manager, Named Executive of the Year by the Diabetes Foundation 2John Timberlake, Valeritas' Executive Vice President and General Manager, Named Executive of the Year by the Diabetes Foundation 3Dr. David Sidransky Appointed Chairman of Champions Biotechnology, Inc. 2Dr. David Sidransky Appointed Chairman of Champions Biotechnology, Inc. 3Dr. David Sidransky Appointed Chairman of Champions Biotechnology, Inc. 4
(Date:10/22/2014)... Inc. (NASDAQ: AWRE ), a leading supplier of ... third quarter ended September 30, 2014. Revenue for ... of 40% compared to $4.3 million in the same quarter ... was $4.1 million compared to $1.0 million in the third ... month period was primarily due to: i) a $1.0 million ...
(Date:10/18/2014)... group of 2,000 patients referred for evaluation of ... molecular diagnosis for 25 percent, including detection of ... mutations contributing to disease, according to a study ... being released to coincide with the American Society ... analyzes the exons or coding regions of thousands ...
(Date:10/17/2014)... German . ... in order to reproduce? And why are there two sexes ... latest issue of the research journal Molecular Human Reproduction ... Ramm from Bielefeld University Bielefeld has compiled this special issue ... a female to copulate with several males in quick succession ...
Breaking Biology News(10 mins):Aware, Inc. Reports Third Quarter 2014 Financial Results 2Aware, Inc. Reports Third Quarter 2014 Financial Results 3Aware, Inc. Reports Third Quarter 2014 Financial Results 4Aware, Inc. Reports Third Quarter 2014 Financial Results 5Aware, Inc. Reports Third Quarter 2014 Financial Results 6Aware, Inc. Reports Third Quarter 2014 Financial Results 7Whole-exome sequencing shows potential as diagnostic tool 2Sperm wars 2
... Lee Lovering, Ph.D., School of Biosciences, University of ... Award for his seminal work on the structural ... and modify cell walls in bacteria. Natalie Strynadka, ... Lovering,s work: "his spectacular abilities in structural biology ...
... 2012) Aeras and the Infectious Disease Research Institute ... trial of IDRI,s novel tuberculosis vaccine candidate, ID93 + ... safety, tolerability and immunogenicity of the vaccine candidate in ... by Johnson County Clin-Trials in Lenexa, Kansas, in close ...
... risks posed to people who work with tiny fibres used ... Research into the health risks posed by nanofibres ... has pinpointed the lengths at which these fibres are ... from a range of materials including carbon, are about 1,000 ...
Cached Biology News:The American Society for Microbiology honors Andrew Lovering 2As TB grows more difficult to control, vaccine candidate to prevent disease enters clinical testing 2
... of modifications of Grace's medium were developed ... and 1970's. Many of these modifications were ... growth of cells derived from the cabbage ... known as TNM-FH. The medium when properly ...
... CLS number is a new ... match Cornings product number. If ... order under the old Sigma-Aldrich ... service for assistance. Mfr ...
... The LIC Duet™ T7•Tag ® ... lac operator, ribosome binding site (rbs), ... T7•Tag coding sequence. The T7•Tag is a ... useful for detection of the target protein ...
... number is a new product ... Cornings product number. If showing ... under the old Sigma-Aldrich number ... for assistance. Mfr Desig: ...
Biology Products: