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:2/17/2017)... 2017  BioGenex, a global leader in molecular ... novel system for quantitative immunohistochemistry (IHC). The system ... Rochester (NY, USA) and Konica-Minolta Inc. ( ... able to accurately quantify the expression of an ... factor receptor-2) in clinical samples. Quantitative IHC is ...
(Date:2/17/2017)... , Feb. 17, 2017  Protagonist Therapeutics, ... data on its oral peptide drug candidates, PTG-100 ... of the European Crohn,s and Colitis Organization (ECCO).  ... Barcelona, Spain from February 15 ... detail preclinical data on Protagonist drug candidates PTG-100 ...
(Date:2/16/2017)...  ImMAGE Biotherapeutics (OTCMKTS: IMMG), an early-stage biotechnology company harnessing ... treatment for triple negative breast cancer (TNBC), announced today their ... program. The YEi Start in ... entrepreneurs grow their business in France ... selected to complete an intensive one week immersion in ...
(Date:2/16/2017)... , Feb. 16, 2017  ArmaGen, Inc., a ... therapies to treat severe neurological disorders, today reported ... with AGT-181, the company,s investigational therapy for the ... as mucopolysaccharidosis type I, or MPS I). The ... (POC) study, presented today at the 13 th ...
Breaking Biology Technology:
(Date:2/3/2017)... -- A new independent identity strategy consultancy firm announces ... Designed to fill a critical niche in technical and ... Mark Crego and Janice Kephart ... expertise that span federal governments, the 9/11 Commission, private ... expertise has a common theme born from a shared ...
(Date:2/1/2017)... Massachusetts , February 1, 2017 IDTechEx ... events on emerging technology, announces the availability of a new report, ... Continue Reading ... ... in industrial and collaborative robots. Source: IDTechEx Report "Sensors for Robotics: ...
(Date:1/26/2017)... , Jan. 26, 2017  Acuity Market ... for Biometrics and Digital Identity".  Acuity characterizes 2017 ... identity when increased adoption reflects a new understanding ... "Biometrics and digital identity are often ... Maxine Most , Principal of Acuity Market ...
Breaking Biology News(10 mins):