Location: Tick and Biting Fly ResearchTitle: Evaluations of methods for the isolation of high quality RNA from bovine and cervine hide biopsies for use in gene expression studies) Author
|Pruett Jr, John|
Submitted to: Journal of Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2012
Publication Date: N/A
Citation: Interpretive Summary: The skin response of a host to a blood-feeding ectoparasite is a critical juncture to investigate in the host-parasite interaction. As the immediate interface between a large animal host and ectoparasites such as ticks, skin punch biopsies taken at the site of tick attachment can provide key information about the host cellular and immune responses that are occurring, and molecular tools enable the evaluation of genes expressed in the skin that are either upregulated or downregulated in response to ectoparasite exposure. Gene expression analyses require high quality nucleic acid (RNA) isolations from the skin, but the complexity of ruminant skin has the propensity to affect successful isolations. Since large animal ruminant studies are costly to replicate and poor quality RNA can negatively impact the reliability of data obtained, the objective of this work was to evaluate storage and RNA isolation methods that would provide optimal template for gene expression analyses. High quality RNA was obtained from both bovine and cervine skin punch biopsies using a TRI Reagent isolation method, suggesting the usefulness of this method for obtaining intact nucleic acids suitable for gene expression studies from other ruminant species.
Technical Abstract: Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA were compared and the use of 4 M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best method for RNA isolation from bovine hide biopsies was then tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrometry and chip electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent® method were again significantly higher than with the other technique, confirming it as the superior method. The TRI Reagent® isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining intact nucleic acids suitable for gene expression studies from other ruminant species.