Data from: A whole-transcriptome approach to evaluating reference genes for quantitative gene expression studies: a case study in Mimulus

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While quantitative PCR (qPCR) is widely recognized as being among the most accurate methods for quantifying gene expression, it is highly dependent on the use of reliable, stably expressed reference genes. With the increased availability of high-throughput methods for measuring gene expression, whole-transcriptome approaches may be increasingly utilized for reference gene selection and validation. In this study, RNA-seq was used to identify a set of novel qPCR reference genes and evaluate a panel of traditional “housekeeping” reference genes in two species of the evolutionary model plant genus Mimulus. More broadly, the methods proposed in this study can be used to harness the power of transcriptomes to identify appropriate reference genes for qPCR in any study organism, including emerging and nonmodel systems. We find that RNA-seq accurately estimates gene expression means in comparison to qPCR, and that expression means are robust to moderate environmental and genetic variation. However, measures of expression variability were only in agreement with qPCR for samples obtained from a shared environment. This result, along with transcriptome-wide comparisons, suggests that environmental changes have greater impacts on expression variability than on expression means. We discuss how this issue can be addressed through experimental design, and suggest that the ever-expanding pool of published transcriptomes represents a rich and low-cost resource for developing better reference genes for qPCR.,Mimulus luteus transcript IDs and sequencesMluteus_transcript_IDs_and_sequences.faMimulus guttatus transcript IDs and sequencesMguttatus_transcript_IDs_and_sequences.faJS1M. luteus var. luteus inbred line EY7 - petal transcriptome "T2" - sample JS1JS2M. guttatus inbred line IM767 - petal transcriptome "T2" - sample JS2JS3M. guttatus inbred line IM767 - leaf transcriptome "T2" - sample JS3JS4M. luteus var. luteus inbred line EY7 - calyx transcriptome "T2" - sample JS4JS5M. guttatus inbred line IM767 - stem transcriptome "T2" - sample JS5JS6M. luteus var. luteus inbred line EY7 - leaf transcriptome "T2" - sample JS6JS7M. guttatus inbred line IM767 - calyx transcriptome "T2" - sample JS7JS8M. luteus var. luteus inbred line EY7 - leaf transcriptome "T2" - sample JS8JS9M. luteus var. luteus inbred line EY7 - stem transcriptome "T2" - sample JS9Mg_T1Mimulus guttatus inbred line CG - transcriptome T1 - complete annotated transcriptomeMimulus_guttatus_complete_w_single_exons_standard_renamed_genes.gff.zipMl_T1Mimulus luteus var. luteus inbred line EY7 - transcriptome T1 - complete annotated transcriptomeMimulus_luteus_complete_w_single_exons_standard_renamed_genes.gff.zipMg_T1_RPKMM. guttatus inbred line CG - transcriptome T1 - RPKM expression report plus raw countsMimulus_guttatus_RPKM_Expression_Report_plusRawCounts.txtMl_T1_RPKMM. l. luteus inbred line EY7 - transcriptome T1 - RPKM expression report plus raw countsMimulus_luteus_RPKM_Expression_Report_plusRawCounts.txt,



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