Navigation Links
Isolate Total RNA and Protein From the Same Sample

mirVana PARIS Kit--Optimized for Recovery of siRNA and miRNA


Small RNA, mRNA, and Protein

Ambion's mirVana PARIS Kit combines the technological advances from both the mirVana miRNA Isolation Kit, optimized for small RNA recovery, and the PARIS (Protein And RNA Isolation System) Kit, specifically developed to isolate protein and RNA from the same experimental sample (see MicroRNAs: Isolation to Functional Analyses). The result is a versatile kit that provides quantitative recovery of native protein and all RNA species, including siRNA and miRNAs. The kit is ideal for downstream assessment of protein and mRNA expression levels, analysis of microRNA (miRNA) expression patterns, and correlation of siRNA expression with protein and mRNA knockdown in RNAi experiments [1].


Reproducible, Quantitative Recovery of Protein and RNA

There is a direct relationship between the inefficient recovery of 5S rRNA and the loss of smaller RNA species, such as miRNAs and siRNAs, during total RNA isolation [2]. For example, total RNA samples prepared with an RNA isolation procedure not optimized for small RNA recovery lack RNA species smaller than ~200 nt, including 5S rRNA, tRNA, and miRNA (Figure 1, PARIS lane). Even with the sensitivity of the mirVana miRNA Detection Kit [2, 3], miR-16 miRNA could barely be detected in 1 g of total RNA isolated from HeLa cells in this way. In contrast, RNA isolated using both the mirVana miRNA Isolation Kit and mirVana PARIS Kit procedures included these small RNA species.

Figure 1. Differential Recovery of Small RNAs. Total RNA was isolated from 1x106 HeLa cells with the indicated kit as per protocol. One g of total RNA was resolved on a 15% denaturing acrylamide gel and stained with EtBr (top) or analyzed by solution hybridization assay with the mirVana miRNA Detection Kit (Ambion) and a probe specific for miR-16 prepared by in vitro transcription with the mirVana Probe Construction Kit (Ambion). Gel was exposed for 6 (middle) or 24 (bottom) hours at -80C


Measuring Reproducibility

Variation in protein, mRNA, and/or small RNA recovery can be an issue if precise quantitation is required in downstream experiments (e.g. RNAi experiments, miRNA profiling). Therefore, reproducible, quantitative recovery of RNA and protein is critical. To determine the reproducibility of the mirVana PARIS procedure, both RNA and protein were isolated from replicates of two types of biological samples, and various quantitative and qualitative parameters were measured. The experimental samples tested were human cell lines assayed 2 days after plating (12 replicates, Figure 2), and mouse liver tissue kept frozen for 2 months at -80C prior to assay (6 replicates, Figure 3). For each experiment, total RNA and protein yields were determined, and samples were assessed for effi cient recovery of long RNA species (real time RT-PCR), small RNA (gel or Northern blot), and protein (Western blot). The results showed that high quality total RNA and protein were efficiently recovered from both samples types. No apparent degradation was observed by gel analysis or Western blot. The mirVana PARIS isolation procedure was also extremely reproducible as each independent replicate contained the same relative proportion of long RNA (18S rRNA, GAPDH, and/or Cyclin D1 mRNA), small RNA (5S rRNA), and protein (GAPDH). Quantitative analysis by real time PCR showed variations of less than 10% (Figure 4).

Figure 2. Analysis of 12 Human Cultured Cell Replicates. HeLa cells were plated at 200,000 cells per well in 6 well plates. Total RNA and protein were isolated 2 days after plating with the mirVana PARIS Kit. One g of total RNA was analyzed on a 15% denaturing polyacryl-amide gel stained with EtBr (left top). Ten g of total protein was resolved on a 10% SDS polyacrylamide gel and analyzed by Western blot with an anti-GAPDH antibody (Ambion) (left bottom). Real time PCR reactions were performed in duplicate using the indicated gene specific primers in combination with SYBR green technology (right).

Figure 3. Analysis of 6 Mouse Liver Replicates. Total RNA and protein were isolated with the mirVana PARIS Kit from 50-80 mg pieces of liver dissected from 6 different mice and kept frozen at -80C for 2 months. One g of total RNA was analyzed on a 1.2% denaturing glyoxal agarose gel stained with EtBr or resolved on a 15% denaturing polyacrylamide gel and analyzed by Northern blot with a 5S rRNA pr obe 5' labeled and purified with the mirVana Probe & Marker Kit (Ambion). GAPDH protein levels were analyzed as in Figure 2. Real time PCR reactions were performed in quadruplicate using TaqMan primer and probe sets for 18S rRNA and GAPDH mRNA (Applied Biosystems).

Figure 4. Summary of Real Time PCR Results. Average Ct values, the range of Ct values, and standard deviations are given for the replicate samples used in real time RT-PCR experiments in Figures 2 and 3.


Powerful System

The mirVana PARIS Kit is a robust method ideal for quantification of protein and RNA, including small RNA species. The kit contains sufficient reagent for 40 protein and RNA isolations; each purification can accommodate 100 to 107 cells or 1 to 100 mg of tissue. The entire procedure is fast and easy, isolating RNA and proteins in less than 30 min. Also, the mirVana PARIS Kit is compatible with samples treated with RNAlater (Ambion), a reagent that stabilizes RNA within tissue prior to processing. For more information about the mirVana PARIS Kit and other tools dedicated to small RNA analysis, please visit www.ambion.com/miRNA.


back to top


Ordering Information
Cat# Product Name Size 1556 mirVana PARIS Kit up to 40 purifications
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture Microdissection
2. Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit
3. A Better Way to Isolate RNA AND Protein From the Same Sample
4. Isolate It All: siRNA miRNA Total RNA Native Protein
5. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
6. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
7. High-Throughput Isolation of Total RNA
8. Fast Isolation of Total RNA from Small Numbers of Cells
9. Total RNA Isolation Protocol 1,2,3
10. Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue
11. Total RNA from Whole Blood for Expression Profiling
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... , June 27, 2016   Ginkgo Bioworks ... to industrial engineering, was today awarded as one ... selection of the world,s most innovative companies. Ginkgo ... scale for the real world in the nutrition, ... engineers work directly with customers including Fortune 500 ...
(Date:6/24/2016)... (PRWEB) , ... June 24, 2016 , ... Researchers at ... most commonly-identified miRNAs in people with peritoneal or pleural mesothelioma. Their findings are the ... read it now. , Diagnostic biomarkers are signposts in the blood, lung fluid ...
(Date:6/23/2016)... , June 23, 2016 /PRNewswire/ - FACIT ... Ontario biotechnology company, Propellon Therapeutics ... development and commercialization of a portfolio of first-in-class ... Epigenetic targets such as WDR5 represent an exciting ... significantly in precision medicine for cancer patients. Substantial ...
(Date:6/23/2016)...  The Prostate Cancer Foundation (PCF) is pleased to announce 24 ... for prostate cancer. Members of the Class of 2016 were selected from a ... Read More About the Class of 2016 PCF Young Investigators ... ... ...
Breaking Biology Technology:
(Date:6/22/2016)... ANGELES , June 22, 2016 /PRNewswire/ ... identity management and verification solutions, has partnered ... edge software solutions for Visitor Management, Self-Service ... provides products that add functional enhancements ... partnership provides corporations and venues with an ...
(Date:6/16/2016)... SAN FRANCISCO , June 16, 2016 /PRNewswire/ ... Market size is expected to reach USD ... report by Grand View Research, Inc. Technological proliferation ... and banking applications are expected to drive the ... ) , The development of ...
(Date:6/7/2016)... June 7, 2016  Syngrafii Inc. and San ... relationship that includes integrating Syngrafii,s patented LongPen™ eSignature ... This collaboration will result in greater convenience for ... union, while maintaining existing document workflow and compliance ... ...
Breaking Biology News(10 mins):