Conservation Genetics Resources

, Volume 4, Issue 1, pp 27–29

Optimization of RNA isolation and leukocyte viability in canid RNA expression studies

  • Marina Oster
  • John P. Pollinger
  • Daniel R. Stahler
  • Robert K. Wayne
Technical Note

DOI: 10.1007/s12686-011-9465-9

Cite this article as:
Oster, M., Pollinger, J.P., Stahler, D.R. et al. Conservation Genet Resour (2012) 4: 27. doi:10.1007/s12686-011-9465-9

Abstract

For RNA transcript analysis, RNA source and quality influence the resolution and representation of true gene expression. We evaluate collection and purification methodologies for isolating gray wolf (Canis lupus) mRNA from peripheral whole blood to maximize detection and quantification of low expression genes. We specifically examine ambient-temperature stabilization of RNA in whole blood at time of collection, subsequent isolation and RNA stabilization of leukocytes to remove globin RNA, and removal of ribosomal RNA (rRNA) using commercially available kits. Ambient temperature RNA stabilization and globin RNA removal were successful with robust mRNA extraction yields and quality, verified by quantitative (q)PCR of reference housekeeping genes. Rhodopsin gene mRNA, present in low levels in peripheral blood, was only detected by qPCR when leukocyte mRNA was additionally treated for ribosomal RNA removal.

Keywords

RNA isolationLeukoctyesRibosomal-DepletionExpressionCanids

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Marina Oster
    • 1
  • John P. Pollinger
    • 2
  • Daniel R. Stahler
    • 2
    • 3
  • Robert K. Wayne
    • 2
  1. 1.Department of BiologyStanford UniversityStanfordUSA
  2. 2.Ecology and Evolutionary Biology DepartmentUniversity of California, Los AngelesLos AngelesUSA
  3. 3.Yellowstone Wolf ProjectYellowstone Center for ResourcesYellowstone National ParkUSA