A Whole-Transcriptome Approach to Evaluating Reference Genes for Quantitative Gene Expression Studies: A Case Study in Mimulus

Abstract

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.

The regulatory network for petal anthocyanin pigmentation is shaped by the MYB5a/NEGAN transcription factor in Mimulus

“…(A) Alignment of MYB5a , MYB5b , and MYB4 . Amino acid sequences were inferred by translating DNA sequences, obtained from the M. l. luteus genome and transcriptome ( Edger et al 2017 ; Stanton et al 2017 ) and from PCR and Sanger sequencing of M. l. variegatus . Black rectangles identify putative Subgroup 6 motifs.…”

“…RNA extractions, cDNA synthesis, and endpoint RT-PCR were performed as described in the Expression Analyses section, to evaluate the expression of the target gene relative to an untransformed control plant. Two different primer pairs targeting MYB5a were used, 33 F-40R and 44 F-45R, along with primers to reference gene GAPDH ( Stanton et al 2017 ) as a positive control (Supplementary Table S1).…”

Selection of Reference Genes for Expression Analysis in Chinese Medicinal Herb Huperzia serrata

“…In the current study, four housekeeping genes ( GAPDH, actin, tubulin , and 18S ) and other four genes ( TBP, SAM, HSP90 , and MUB ) were used as query genes for the blast against the RNA-seq data (Yang et al, 2017) to find the homologous genes (Stanton et al, 2017). Genes with similar expression levels in four different tissues were selected as candidates as previously reported (Tan et al, 2015).…”

Selection of reference genes for flowering pathway analysis in the masting plants, Celmisia lyallii and Chionochloa pallens, under variable environmental conditions

“…There are numerous reports of selection and validation of reference genes in model plant species including Arabidopsis thaliana 20 , Oryza sativa 30 , Helianthus annuus 31 , Triticum aestivum 32,33 , Zea mays 34 and Glycine max 35 . The list also includes diverse non-model plant species including flax 36 , white clover 37 , bamboo 38 , peach 39 , Mimulus 40 , watermelon 41 , grape vine 42 , and lettuce 43 . However, in most of these cases the selection of suitable reference gene(s) was based on tightly controlled experimental conditions, whereas it has been reported that the expression of these gene(s) can vary in different tissues under different experimentally controlled conditions 26,44–48 .…”