Primer design and transcript quantification of a highly multiplexed RT-PCR for a nonmodel avian species

Multiplexed qRT‐PCR assays are currently lacking for nearly all species without genome or transcriptome resources. Here, we present a strategy for primer design of highly multiplexed qRT‐PCR assays, evaluate Beckman Coulter’s Quant Tool gene expression quantification software and provide details of our assay for the North American songbird Carpodacus mexicanus (house finch), for which only small sections of genome sequence are available. We combined Beckman Coulter’s eXpress Designer module for creating custom multiplex primers with the free, online program Amplify 3 to design and evaluate primers computationally before testing them empirically. We also generated a standard curve for each gene included in the final multiplex. We compared models using cubic and quadratic polynomial estimators that did and did not force the intercept through zero. Ultimately, we used the sequences available for 316 clones differentially expressed in cDNA macroarray and microarray comparisons, and from these sequences, we were able to generate a set of transcript‐specific primers for use with the GeXP analyser for 20 house finch genes.