Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection

MicroRNAs (miRNAs) are a class of small, endogenously encoded regulatory RNAs that function to post-transcriptionally regulate gene expression in a wide variety of eukaryotes. Within organisms, some mature miRNAs, such as paralogous miRNAs, have nearly identical nucleotide sequences, which makes the...

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Bibliographic Details
Published inRNA (Cambridge) Vol. 16; no. 6; pp. 1268 - 1274
Main Authors Zeiner, Gusti M, Boothroyd, John C
Format Journal Article
LanguageEnglish
Published United States Cold Spring Harbor Laboratory Press 01.06.2010
Subjects
Animals
Base Pairing - genetics
Base Sequence
DNA Primers
Gene Expression Regulation
Humans
Method
MicroRNAs - genetics
Oligonucleotide Array Sequence Analysis
Ribonucleases - metabolism
RNA, Messenger - genetics
RNA, Protozoan - genetics
Sequence Alignment
Sequence Homology
Templates, Genetic
Toxoplasma - genetics
Toxoplasma - pathogenicity
Toxoplasmosis - genetics
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Summary:MicroRNAs (miRNAs) are a class of small, endogenously encoded regulatory RNAs that function to post-transcriptionally regulate gene expression in a wide variety of eukaryotes. Within organisms, some mature miRNAs, such as paralogous miRNAs, have nearly identical nucleotide sequences, which makes them virtually indistinguishable from one another by conventional hybridization-based approaches. Here we describe two inexpensive, sensitive methods for rapidly discriminating between paralogous miRNAs or other closely related miRNAs and for quantifying their abundance. The first approach is a sequential ribonuclease-protection and primer-extension assay; the second approach is a primer-extension assay that employs short oligonucleotide probes to exacerbate the instability of mismatched probe:miRNA hybrids. Both approaches are rapid and can be easily performed in their entirety using common laboratory equipment. As a proof of concept, we have used these methods to determine the exact identities of the human miR-17 family members that are increased by infection with the intracellular parasite Toxoplasma gondii. These methods can be used to rapidly and inexpensively discriminate between any closely related miRNAs in any organism.
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Reprint requests to: John C. Boothroyd, Department of Microbiology and Immunology, Fairchild Science Building, Room D305, 299 Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5124, USA; e-mail: john.boothroyd@stanford.edu; fax: (650) 725-6757.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.2069310