target validation

A Proteomics Approach to Validating microRNA Targets

by Chris on September 16, 2010

from Nature Asia-Pacific.com

A sensitive and reproducible method to validate several hundred microRNA target genes in one experiment is described online this week in Nature Methods. These results give a better understanding of how these target genes could be regulated.

MicroRNAs are short RNA molecules that do not encode a protein but are crucial in regulating gene expression by blocking translation or marking the RNA for degradation. The targets of specific microRNAs can be predicted by computational algorithms, but experimental approaches to validate the predicted targets at a large scale are needed.

Michael Hengartner and colleagues report a proteomics approach to follow up on computationally predicted microRNA target genes in the roundworm Caenorhabditis elegans. To determine which genes are regulated by a specific microRNA known as let-7, the researchers compared protein levels in normal worms to protein levels in mutant worms in which let-7 levels were reduced. They used a highly sensitive detection technique called selected reaction monitoring (SRM) mass spectrometry in combination with a quantitative method, isotope-coded affinity tagging (ICAT). When the protein levels changed in the mutant worms compared to the normal worms, this indicated that the gene was indeed regulated by let-7.

The general approach should be readily adaptable to validate the predicted target genes of any microRNA in any organism. (read more… )

Jovanovic M, Reiter L, Picotti P, Lange V, Bogan E, A Hurschler B, Blenkiron C, J Lehrbach N, C Ding X, Weiss M, P Schrimpf S, A Miska E, Großhans H, Aebersold R, O Hengartner M. (2010) A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans. Nat Methods [Epub ahead of print].  [abstract]

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New microRNA Target Validation Protocol

by Chris on September 24, 2009

Validating microRNA Target Transcripts Using Zebrafish Assays

By: Luke Pase, Graham J. Lieschke

Hundreds of tiny noncoding RNAs known as microRNAs (miRNAs) have been identified in the genomes of plants and animals. Studies are increasingly demonstrating that individual miRNAs are important in normal development and physiology. miRNAs are regulators of gene expression that bind target mRNAs and modulate their translation and turnover. The specificity of this regulation is achieved by partial sequence complementarity between the miRNA and its target mRNA. Understanding which mRNAs are targeted by each particular microRNA is critical to an understanding of the biologic role of any particular miRNA. Bioinformatic approaches can be used to predict mRNAs that may be miRNA targets, but each of these predictions requires experimental validation. We describe a method for a reporter assay based on a fluorescence intensity readout that uses transient techniques in zebrafish to easily deliver the reporter assay components. In addition, we describe a rigorously controlled strategy for determining the bona fide miRNA binding sites in the 3′UTR of mRNAs. (read more)

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