Abstract
Differential gene expression is a key factor driving phenotypic divergence. Determining when and where gene expression has diverged between organisms requires a quantitative method. While large-scale approaches such as microarrays or high-throughput mRNA sequencing can identify candidates, quantitative RT-PCR is the definitive method for confirming gene expression differences. Here, we describe the steps for performing qRT-PCR including extracting total RNA, reverse-transcribing it to make a pool of cDNA, and then quantifying relative expression of a few candidate genes using real-time or quantitative PCR.
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Acknowledgments
The author would like to thank Tyrone Spady for help with the development of the multigene construct for measuring PCR efficiencies and Christopher Hofmann for helpful comments to improve this manuscript. This work was supported with funding from NSF (IOS-0841270).
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Carleton, K.L. (2012). Quantification of Transcript Levels with Quantitative RT-PCR. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_17
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DOI: https://doi.org/10.1007/978-1-61779-228-1_17
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