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Chimeric RNA pp 109-116 | Cite as

RNase Protection Assay

  • Jianzhu Zhao
  • Jun Tang
  • Justin Elfman
  • Hui LiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2079)

Abstract

Unbound, single-stranded RNA can be digested by RNase (A or T1) to ribonucleotides, whereas double-stranded RNA is not digested by RNase. Based on this principle, the RNase Protection Assay (RPA) is used to validate chimeric RNAs. Importantly, this assay does not employ reverse transcription (RT), thus avoiding potential false-positive results which could occur during RT such as template-switching. We first generate RNA probes with 32phosphate (P) or biotin that are complementary to the predicted nucleotide sequence of the chimeric RNA, then hybridize them to RNA samples. The labeled RNA probes can bind specifically with the target chimeric RNA in order to form double-stranded RNA. This newly formed RNA is resistant to digestion by RNase and therefore can be identified by high-resolution, denaturing polyacrylamide gel electrophoresis.

Key words

Chimeric RNA RNase Double-stranded RNA Single-stranded RNA Radioactively labeled Denaturing polyacrylamide gel electrophoresis 

References

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of OncologyShengjing Hospital of China Medical UniversityShenyangChina
  2. 2.Department of Thoracic SurgeryShengjing Hospital of China Medical UniversityShenyangChina
  3. 3.Department of Biochemistry and Molecular Genetics, School of MedicineUniversity of VirginiaCharlottesvilleUSA
  4. 4.Department of Pathology, School of MedicineUniversity of VirginiaCharlottesvilleUSA

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