Navigation Links
RNase Protection Assay (RPA),,,Using DIG-Labeled RNA Probes

The ribonuclease protection assay (RPA) is a highly sensitive and specific method for the detection and quantitation of mRNA species. The assay was made possible by the discovery and characterization of bacteriophageencoded DNA-dependent RNA polymerases (SP6, T7, and T3). These enzymes possess several properties that make them well suited for the preparation of highly specific hybridization probes. They are very specific in their action, recognizing only phage-specific promoter sequences that are not part of other DNA sequences.

To generate a probe for use in this assay, it is necessary to subclone a fragment that contains the cDNA sequences of interest downstream of one of the mentioned phage promoters. The orientation of the insert must direct the synthesis of complementary (anti-sense) RNA. Ideally, this construct should be digested with a restriction enzyme (RE), which produces 5 overhangs, to create a linear template that will be transcribed into a 100 to 300-base run-off transcript.

The RPA technique is based on the hybridization in solution of a labeled anti-sense probe RNA with the RNA pool being tested. Complementary portions of labeled probe and mRNA from the pool to be analyzed form hybrids that are resistant to digestion with single strandspecific RNase T1 and RNase A. Because of its sensitivity, the RPA can, in general, be performed on total RNA preparations derived by standard methods from either frozen tissues or cultured cells, without further purification of polyA+ RNA.

Following treatment with ribonuclease, the RNase-protected fragments are resolved on a denaturing polyacrylamide or agarose gel. Subsequent detection reveals the presence of target mRNA in t he sample by the appearance of an appropriately sized probe fragment.

Factors Influencing the Assay

Transcript quality
  • Long transcripts tend to give incomplete transcribed probes. We suggest the use of a relatively small probe for this assay (100300 nucleotides).
  • RNase contamination
  • Presence of transcription-termination signals in the template.
  • Wraparound transcripts resulting from the use of REs that produce either a 3 overhang or a blunt end. It is strongly recommended to use a RE that produces a 5 overhang.

Quality of target RNA
  • RNase contamination: When probe fragmentation is observed in the protection assay, check the integrity of your RNA sample by agarose electrophoresis.
  • DNA contamination

Amount of sample and labeled probe

The sample size is usually in the milligram-range and the amount of labeled probe is around 500 pg1,000 pg.





'"/>

Source:


Page: All 1 2

Related biology technology :

1. Protector RNase Inhibitor
2. Protector RNase Inhibitor Enhance the Protection of RNA against Degradation
3. Inducing RNAi with siRNA Cocktails Generated by RNase III
4. Dicer vs. RNase III for Preparation of siRNA Cocktails
5. Combat RNase Contamination in the Lab
6. Measuring RNase Activity A Real-time Kinetic Analysis
7. RNase Activity in Mouse Tissue: Classification, Hierarchy, and Methods for Control
8. Nuclease Protection Assays: The Basics
9. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
10. Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" )
11. Organize Your Data from the Big Blue Mutation Assay
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/25/2017)... ... April 25, 2017 , ... ... supplier of Common Lisp (CL) development tools, and market leader for Semantic ... key performance enhancements now available within the most effective system for developing and ...
(Date:4/25/2017)... ... April 25, 2017 , ... Covalent Metrology has ... Services unit provides high-quality data to clients, both faster and cheaper than ... There are no price premiums, and customers are welcome to participate in the ...
(Date:4/21/2017)... ... 21, 2017 , ... The 2017 Colorado Manufacturing Awards celebrates ... nominees and well as the first-year award for 2017 Manufacturing Women of the ... hosted by CompanyWeek and Manufacturers Edge, among other sponsors. , The Colorado ...
(Date:4/21/2017)... ... ... The AMA is happy to announce that $48,000 in scholarships will be awarded ... created through funds donated by model aviation organizations and individuals, AMA members, and a ... Committee, which is made up of model aviation pilots and enthusiasts. The committee evaluates ...
Breaking Biology Technology:
(Date:3/23/2017)... 23, 2017 Research and Markets has announced ... & Trends - Industry Forecast to 2025" report to their ... The ... CAGR of around 8.8% over the next decade to reach approximately ... the market estimates and forecasts for all the given segments on ...
(Date:3/20/2017)... March 20, 2017 PMD Healthcare announces the ... and Wellness Management System (WMS), a remote, real-time lung ... 2010, PMD Healthcare is a Medical Device, Digital Health, ... dedicated to creating innovative solutions that empower people to ... intent focus, PMD developed the first ever personal spirometer, ...
(Date:3/7/2017)... England , March 7, 2017 Brandwatch , ... chosen by The Prince,s Trust to uncover insights to ... across The Trust. The UK,s leading youth charity will ... social campaign results and get a better understanding of the topics ... ...
Breaking Biology News(10 mins):