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Open Access Highly Accessed Research article

RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Christopher RJ Laver and John S Taylor*

Author Affiliations

University of Victoria, Department of Biology, Victoria, British Columbia, Canada

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BMC Evolutionary Biology 2011, 11:81  doi:10.1186/1471-2148-11-81

Published: 29 March 2011

Additional files

Additional file 1:

Spectral characteristics of guppy light-dark cycle aquaria. Illumination was provided by broad spectrum fluorescent bulbs (GE F32T8SP/65K), with relative spectral irradiance measured by a USB2000 spectrophotometer (Ocean Optics, Inc.) at the air to water boundary during the light phase.

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Additional file 2:

Cross-amplification controls for LWS primer specificity. (A) Locus-specific primer (LSP) sets, detailed in the methods, for each LWS (A180, P180, S180, and S180r) were individually used in an attempt to amplify off of LWS-containing plasmids (1.0E07 copies/reaction) under the RT-qPCR conditions detailed in the methods. Although cloned opsin gene (pGEM::LWS) sequences are not full length (i.e., incomplete open reading frame, ORF) their respective primer site locations are shared (although different in sequence) among all four genes. Plasmid inserts were generated using primers shown in part C. Amplification and dissociation curves are shown with corresponding agarose gel-electrophoresis images of resultant amplicons. 1kb+ DNA ladder was used (Invitrogen®). All reactions were run in parallel. Note the gel image corresponding to the S180r primer panel is not the same dimensions as the others, as lane gaps were not used during gel loading. (B) NAC and NTC controls are shown. (C) Table of Oligonucleotide primers and sequences used to generate LWS plasmids for cross-amplification experiments.

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Additional file 3:

Amplification efficiency differences among visual opsins. qPCR plasmid standard-curve values used to assess differences in amplification efficiencies among six of the 10 visual-opsin gene constructs.

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Additional file 4:

Reference gene copy number for primary (A) and secondary (B) surveys. Transcript copy number of COI, β-actin, and Myosin-HC reference genes determined by RT-qPCR analysis of guppy cDNA samples. (L) and (R) denote left and right eyes, respectively. Error bars (±S.E.M. of triplicate reactions) are shown for all samples, though most are too small to see.

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Additional file 5:

Normalization to multiple reference genes. Justification of reference genes used for normalization.

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