Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.     

Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR

Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.     

Reference Gene Selection for Real-Time Quantitative PCR Analysis of the Mouse Uterus in the Peri-Implantation Period

The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm^PLUS, NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels.     

Evaluation of potential reference genes for qRT-PCR data normalization in HeLa cells

Reference genes selection is one of the most important stages in qPCR data normalization when a problem of quantitative determination of gene expression is addressed. Stability of gene expression level in all experimental conditions is a basic criterion for the reference gene selection. Over the past decade a lot of publications concerning validation methods of suitable reference genes appeared. In this paper, the main approaches (ΔCt, geNorm, qBase and Haller’s equivalence test) were applied for the reference genes identification in HeLa cell line which is one of the most popular cellular models. Expression stability of seven candidate genes (HPRT1, ACTB, GAPDH, RPS18, HSPC3, UBC and SDHA) was determined at standard conditions, under heat shock and during relaxation. The genes RPS18 and HSPC3 were chosen as reference after the combination of all the validation methods.     

Selection of reference genes for quantitative real-time PCR in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.     

Identification and Validation of Reference Genes for Quantification of Target Gene Expression with Quantitative Real-time PCR for Tall Fescue under Four Abiotic Stresses

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.     

Transient expression assays in grapevine: a step towards genetic improvement

In the past few years, the usefulness of transient expression assays has continuously increased for the characterization of unknown gene function and metabolic pathways. In grapevine (Vitis vinifera L.), one of the most economically important fruit crops in the world, recent systematic sequencing projects produced many gene data sets that require detailed analysis. Due to their rapid nature, transient expression assays are well suited for large‐scale genetic studies. Although genes and metabolic pathways of any species can be analysed by transient expression in model plants, a need for homologous systems has emerged to avoid the misinterpretation of results due to a foreign genetic background. Over the last 10 years, various protocols have thus been developed to apply this powerful technology to grapevine. Using cell suspension cultures, somatic embryos, leaves or whole plantlets, transient expression assays enabled the study of the function, regulation and subcellular localization of genes involved in specific metabolic pathways such as the biosynthesis of phenylpropanoids. Disease resistance genes that could be used for marker‐assisted selection in conventional breeding or for stable transformation of elite cultivars have also been characterized. Additionally, transient expression assays have proved useful for shaping new tools for grapevine genetic improvement: synthetic promoters, silencing constructs, minimal linear cassettes or viral vectors. This review provides an update on the different tools (DNA constructs, reporter genes, vectors) and methods (Agrobacterium‐mediated and direct gene transfer methods) available for transient gene expression in grapevine. The most representative results published thus far are then described.