title: Quantification of transcript levels with quantitative RT-PCR.
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.
authors: University of Maryland, College Park, MD, USA. kcarleto@umd.edu
authors: Karen L
authors: KL
authors: Carleton
mesh_terms: D000818:Animals; D005453:Fluorescence; D055355:Opsins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription / Q000235:genetics; D013223:Statistics as Topic; D013698:Templates, Genetic
chemical_list: D055355:Opsins; D012333:RNA, Messenger