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Author Notes:

Wendy M. Zinzow-Kramer, 1510 Clifton Rd, Rollins Research Center, Rm 2006, Emory University, Atlanta, GA 30322, Phone: +1-404-727-5879, Fax: +1-404-727-4034, wzinzow@emory.edu

We are grateful to Laura Carruth for providing zebra finches; and to the Emory University Departments of Biology and Human Genetics for use of resources.

We thank Jake Metzler and the Forest Society of Maine for access to our field site, the Hemlock Stream Forest.

We are obliged to the University of Maine School of Biology and Ecology and Clarissa Henry for logistical support in Maine.

We thank Demesew Abebe for technical assistance and Adela Annis; Elaina Burns; Jenna Cava; Allison Cornell; Chris Gurguis; Clifton McKee; and Justin Michaud for field assistance.

Subject:

Research Funding:

This work was supported by NIMH 1R01MH082833-01A2 to DLM; and NSF SMA-1306132 to WZK.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Behavioral Sciences
  • Endocrinology & Metabolism
  • Housekeeping gene
  • Quantitative real-time PCR (qPCR)
  • Reference gene
  • Songbird
  • White-throated sparrow
  • Zebra finch
  • WHITE-THROATED SPARROW
  • MESSENGER-RNA EXPRESSION
  • ZONOTRICHIA-LEUCOPHRYS-GAMBELII
  • MALE ZEBRA FINCHES
  • SOCIAL-BEHAVIOR
  • HOUSE SPARROWS
  • CHROMOSOMAL-POLYMORPHISM
  • HOUSEKEEPING GENES
  • PASSER-DOMESTICUS
  • ALBICOLLIS GMELIN

Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds

Tools:

Journal Title:

Hormones and Behavior

Volume:

Volume 66, Number 2

Publisher:

, Pages 267-275

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.

Copyright information:

© 2014 Elsevier Inc. All rights reserved. CC BY NC ND 4.0

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