On measuring miRNAs after transient transfection of mimics or antisense inhibitors

The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manip...

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Bibliographic Details
Published in:PloS one 2013-01, Vol.8 (1), p.e55214-e55214
Main Authors: Thomson, Daniel W, Bracken, Cameron P, Szubert, Jan M, Goodall, Gregory J
Format: Article
Language:English
Subjects:
Antisense RNA
Argonaute Proteins - genetics
Base Sequence
Biology
Cancer
Deoxyribonucleic acid
DNA
DNA Primers
Experiments
Fluorescence
Gene expression
Genetic research
Genomes
Inhibition
Inhibitors
Localization
Lysis
Measurement
Medical research
MicroRNA
MicroRNAs
MicroRNAs - analysis
Microscopy
miRNA
Molecular Mimicry
Observations
Overexpression
Pathology
Physiology
Polymerase Chain Reaction
Properties
Ribonucleic acid
RNA
RNA, Antisense - genetics
Target recognition
Transfection
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Summary:The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0055214