A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5' RACE and a molecular beacon probe.

Nucleic Acids Research
Annette LashamGlen Reid

Abstract

Specific detection of mRNA cleavage by 5'RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5'-RNA-linker-mediated RACE (5'-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5'-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. With its sensitivity and specificity, this variation on the 5'RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo.

References

Apr 11, 1993·Nucleic Acids Research·M Fromont-RacineT Grange
Feb 3, 1998·Nature Biotechnology·S TyagiF R Kramer
Mar 1, 1996·Nature Biotechnology·S Tyagi, F R Kramer
Feb 16, 2002·Methods : a Companion to Methods in Enzymology·K J Livak, T D Schmittgen
Sep 17, 2002·Cell·Javier MartinezThomas Tuschl
Nov 13, 2004·Nature·Jürgen SoutschekHans-Peter Vornlocher
Aug 23, 2005·Clinica Chimica Acta; International Journal of Clinical Chemistry·Salvatore A E MarrasFred Russell Kramer
Feb 17, 2006·Molecular Therapy : the Journal of the American Society of Gene Therapy·Mark A Behlke
Mar 28, 2006·Nature·Tracy S ZimmermannIan MacLachlan
May 26, 2006·The Journal of Gene Medicine·Magdolna G SebestyénJon A Wolff
Jul 13, 2006·Annual Review of Biomedical Engineering·Derek M Dykxhoorn, Judy Lieberman
Sep 2, 2006·Nucleic Acids Research·Angus T De SouzaRoger G Ulrich
Sep 22, 2006·Gene Therapy·T SudaD Liu
Dec 15, 2006·Gene Therapy·H Herweijer, J A Wolff
Sep 18, 2007·Nature Biotechnology·Christian WolfrumMarkus Stoffel
Mar 28, 2008·Nature·Mark E KleinmanJayakrishna Ambati
Jun 5, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Louis Doré-SavardPhilippe Sarret
Aug 13, 2008·Proceedings of the National Academy of Sciences of the United States of America·Maria Frank-KamenetskyKevin Fitzgerald
Aug 21, 2008·Human Gene Therapy·Marjorie RobbinsIan MacLachlan
Dec 20, 2008·Oligonucleotides·William QuerbesDinah W Y Sah
Jan 31, 2009·Nature Reviews. Drug Discovery·Kathryn A WhiteheadDaniel G Anderson
Feb 21, 2009·The Journal of Clinical Investigation·Adam D JudgeIan MacLachlan
May 16, 2009·Oligonucleotides·Marjorie RobbinsIan MacLachlan
May 28, 2009·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Lingegowda S MangalaAnil K Sood

❮ Previous
Next ❯

Citations

Jun 2, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Maria C Palanca-WesselsOliver W Press
Nov 22, 2013·PloS One·Cherie BlenkironAnnette Lasham
Nov 13, 2013·Immunology and Cell Biology·Norliana KhairuddinNigel A J McMillan
May 9, 2012·Cellular and Molecular Life Sciences : CMLS·Jeonghyun AhnHeuiran Lee
Jun 21, 2014·Expert Opinion on Drug Delivery·Stefano ColomboCamilla Foged
Dec 15, 2015·The Journal of Infectious Diseases·A Castellanos-GonzalezA C White
Dec 4, 2015·Journal of Basic Microbiology·Lei YangHua Tang
Nov 10, 2011·The Journal of Pathology·Jonathan K Watts, David R Corey
Mar 7, 2014·Thrombosis Research·Maggie L Kalev-ZylinskaChristopher M Ward
Apr 14, 2016·International Journal of Cancer. Journal International Du Cancer·Annette LashamCristin G Print

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
electrophoresis
PCRs

Software Mentioned

MBRACE

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.