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Anal Biochem. 2016 Sep 1;508:65-72. doi: 10.1016/j.ab.2016.06.022. Epub 2016 Jun 24.

A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

Author information

1
University of Minnesota, Integrated BioSciences, 1035 University Drive, SMed 223, Duluth, MN 55812-3031, USA; University of Minnesota Medical School - Duluth, 1035 University Drive, Duluth, MN 55812-3031, USA. Electronic address: veronica.a.seifert@gmail.com.
2
University of Minnesota, Integrated BioSciences, 1035 University Drive, SMed 223, Duluth, MN 55812-3031, USA; University of Minnesota Medical School - Duluth, 1035 University Drive, Duluth, MN 55812-3031, USA. Electronic address: bclarke@d.umn.edu.
3
Peromyscus Genetic Stock Center University of South Carolina Office of Research, Columbia, SC 29208, USA. Electronic address: janetpc@mailbox.sc.edu.
4
University of Minnesota, Integrated BioSciences, 1035 University Drive, SMed 223, Duluth, MN 55812-3031, USA; University of Minnesota Medical School - Duluth, 1035 University Drive, Duluth, MN 55812-3031, USA. Electronic address: ltbemis@d.umn.edu.

Abstract

A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species.

KEYWORDS:

16S mitochondrial ribosomal RNA; Extracellular RNA; High resolution melt; Peromyscus leucopus; Peromyscus maniculatus

PMID:
27349513
DOI:
10.1016/j.ab.2016.06.022
[Indexed for MEDLINE]

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