Validating Internal Control Genes for the Accurate Normalization of qPCR Expression Analysis of the Novel Model Plant Setaria viridis

Abstract

Employing reference genes to normalize the data generated with quantitative PCR (qPCR) can increase the accuracy and reliability of this method. Previous results have shown that no single housekeeping gene can be universally applied to all experiments. Thus, the identification of a suitable reference gene represents a critical step of any qPCR analysis. Setaria viridis has recently been proposed as a model system for the study of Panicoid grasses, a crop family of major agronomic importance. Therefore, this paper aims to identify suitable S. viridis reference genes that can enhance the analysis of gene expression in this novel model plant. The first aim of this study was the identification of a suitable RNA extraction method that could retrieve a high quality and yield of RNA. After this, two distinct algorithms were used to assess the gene expression of fifteen different candidate genes in eighteen different samples, which were divided into two major datasets, the developmental and the leaf gradient. The best-ranked pair of reference genes from the developmental dataset included genes that encoded a phosphoglucomutase and a folylpolyglutamate synthase; genes that encoded a cullin and the same phosphoglucomutase as above were the most stable genes in the leaf gradient dataset. Additionally, the expression pattern of two target genes, a SvAP3/PI MADS-box transcription factor and the carbon-fixation enzyme PEPC, were assessed to illustrate the reliability of the chosen reference genes. This study has shown that novel reference genes may perform better than traditional housekeeping genes, a phenomenon which has been previously reported. These results illustrate the importance of carefully validating reference gene candidates for each experimental set before employing them as universal standards. Additionally, the robustness of the expression of the target genes may increase the utility of S. viridis as a model for Panicoid grasses.

Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis

“…Several studies have attempted to validate the use of a number of ‘traditional’ housekeeper genes, including UBIQUITIN , ACTIN , TUBULIN , GLYCERALDEHYDE 3 - PHOSPHATE DEHYDROGENASE and the ribosomal RNA, 18S - RNA , to normalise gene expression data in Setaria tissues. However, the majority of these ‘traditional’ housekeeper genes returned poor expression stability under differing experimental conditions or across the samples tested, including seedlings, whole-stem, whole-leaf and a leaf developmental gradient [9, 22, 25]. Here, we assessed the suitability of eleven candidate reference genes for the accurate and reliable normalisation of gene expression quantification across thirteen developmentally distinct tissues of S. viridis , including internodes 4, 5 and 6, leaves 4, 5 and 6, inflorescence stems S1, S2 and S3, and the four developmental zones of the expanding internode, internode 5.…”

“…The second dataset analysed, was derived from leaf 3 sampled from a 10 day old plant [5]. The remaining four candidate reference genes selected for detailed assessment in this study, namely CUL , FPGS , PGM and PP2A , were included based on their expression profiles reported in previous RT-qPCR studies performed in S. viridis [9] and sorghum [14]. …”

“…The RT-qPCR approach provides greatly enhanced levels of sensitivity and accuracy for gene expression analysis compared to the more conventional methods, semi-quantitative RT-PCR and northern blot hybridisation [7]. The RT-qPCR approach is reliant upon the exponential incorporation of fluorescent dyes into amplified products [8], and has become the method of choice to validate RNA sequencing (RNA-Seq) data, or to quantify the abundance of a select group of target genes stemming from a large population of expressed transcripts [9, 10]. The accuracy and reliability of gene expression data derived from RT-qPCR experiments is not only influenced by the quality and quantity of the RNA used as template for complementary DNA (cDNA) synthesis, but is highly dependent on normalisation to one or more stably expressed reference genes.…”

“…Several housekeeper genes have been widely used in Arabidopsis thaliana ( CYCLOPHILIN ( CYC ), ACTIN2 ( ACT2 ) and ELONGATION FACTOR 1 α ( EF1 α; [15, 16])), rice ( ACTIN1 ( ACT1 ) and UBIQUITIN5 ( UBI5 ; [7, 11])), and sorghum ( SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ) and EUKARYOTIC INITIATION FACTOR 4α ( EIF4α ; [14])). However, a growing body of research has shown that housekeeper genes cannot be utilised universally across different plant species, experimental conditions, or even across different developmental stages within a single species [9, 11, 14]. Further, normalisation of RT-qPCR data using just a single housekeeper gene is no longer recommended [17, 18], as such an approach could result in a biased or incorrect interpretation of the expression of the studied gene(s).…”

“…The eleven normalisation candidates assessed were identified from two RNA-Seq datasets, namely an elongating internode dataset [4] and an expanding leaf dataset [5], in combination with those reported previously for RT-qPCR analyses in other C 4 species [9, 14]. NormFinder and geNorm analyses of candidate reference gene performance identified three candidates, SERINE / THERONINE - PROTEIN PHOSPHATASE 2A ( PP2A ), 5 ′- ADENYLYLSULFATE REDUCTASE 6 ( ASPR6 ) and DUAL SPECIFICITY PHOSPHATASE ( DUSP ), as the most suitable combination of reference genes for the accurate and reliable normalisation of target gene expression across S. viridis tissues.…”