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

• Type of Material: Article
• Authors: Wendy M. Zinzow-Kramer, Emory University; Brent M. Horton, Emory University; Donna Maney, Emory University
• Language: English
• Date: 2014-07-01
• Publisher: Elsevier: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2014 Elsevier Inc. All rights reserved. CC BY NC ND 4.0
• Final Published Version (URL): http://dx.doi.org/10.1016/j.yhbeh.2014.04.011
• Title of Journal or Parent Work: Hormones and Behavior
• ISSN: 0018-506X
• Volume: 66
• Issue: 2
• Start Page: 267
• End Page: 275
• Grant/Funding Information: This work was supported by NIMH 1R01MH082833-01A2 to DLM; and NSF SMA-1306132 to WZK.
• Keywords: ZONOTRICHIA-LEUCOPHRYS-GAMBELII; Science & Technology; Housekeeping gene; CHROMOSOMAL-POLYMORPHISM; Zebra finch; White-throated sparrow; Quantitative real-time PCR (qPCR); ALBICOLLIS GMELIN; MESSENGER-RNA EXPRESSION; SOCIAL-BEHAVIOR; Reference gene; Songbird; Behavioral Sciences; PASSER-DOMESTICUS; WHITE-THROATED SPARROW; Endocrinology & Metabolism; HOUSEKEEPING GENES; HOUSE SPARROWS; Life Sciences & Biomedicine; MALE ZEBRA FINCHES
• Research Categories: Psychology, General

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.