Selection Of Reference Genes Research Articles

Selection of reference genes for quantitative real-time PCR analysis in exogenous hormone-treated Lycoris aurea.

Selection of reference genes for quantitative real-time PCR analysis in exogenous hormone-treated Lycoris aurea.

Identification of reference genes for gene expression assessment in Avena sativa under biotic stress triggered by Blumeria graminis

A repeatable and reliable reverse transcription quantitative PCR (RT-qPCR) experiment depends upon proper reference genes (RGs) selection. This study aims to examine the expression stability of nine candidate RGs for the Avena sativa – Blumeria graminis experimental setup. B. graminis causes powdery mildew - the most devastating and economically important fungal disease of crops worldwide. RGs were evaluated in Pm3 and Pm4 oat differential lines and the susceptible cultivar Fuchs during compatible and incompatible interactions with different pathotypes of Blumeria graminis f. sp. avenae in six-time points post inoculation. The identification of genes exhibiting high expression stability was done by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt). The results indicated that regardless of the analysed group, two most stable RGs are required for data normalization. The most sufficient RGs combination was HNR (heterogeneous nuclear ribonucleoprotein 27 C) + EIF4A (eukaryotic initiation factor 4 A‑3). ARF (ADP‑ribosylation factor) could also be pondered as demonstrating high expression stability. These genes can be considered universal candidates for RT-qPCR normalization to study interaction with B. graminis as well as Puccinia coronata and Puccinia graminis, as confirmed by our previous research. The worst candidate for data standardisation was TUA (α- tubulin). To our best knowledge, this is the first report regarding RGs’ selection in this pathosystem. Identified RGs are proper normalisation candidates for gene expression studies in the A. sativa infected by B. graminis as well as other related pathogens.

Selection and validation of reference genes for qRT-PCR normalization in dayflower (Commelina communis) based on the transcriptome profiling

BackgroundDayflower (Commelina communis), a widely invasive weed, thrives well under a variety of abiotic stresses, including drought and herbicides, and harms the growth of crops such as maize and soybean. Gene expression in dayflower is an important but understudied area due to the lack of reliable reference genes.ResultsFifteen candidate reference genes, which are common reference genes and homologous to those used in other plants, were selected through RNA-seq datasets of dayflower. The expression stability of these screened reference genes was evaluated under three abiotic stresses (drought, herbicide and copper) and in five organs (roots, stems, leaves, flowers and seeds) using five commonly used software programs (geNorm, NormFinder, BestKeeper, ΔCt and RefFinder). The results showed that API5 and SAND had the highest stability in stems, while SAND, EF1A and API5 had the highest stability in roots. Moreover, SAND, ALDH113 and API5 were most stably expressed under copper stress, and EF1A, SAND and API5 were most stable under drought stress. SAND was consistently the most stably expressed gene in both the organs and all samples. Notably, The SAND gene ranked among the top three in terms of stability in all abiotic treatments and in various organs. This result indicates that the SAND gene is suitable for qRT-PCR experimentation in diverse tissues and under multiple (drought, herbicide and copper) abiotic stress conditions in dayflower.ConclusionThis study identified the most stably expressed reference genes under three abiotic stresses and in five organs of dayflower, and SAND showed high expression stability under various experimental conditions, making it a reliable reference gene for gene expression analysis experiments under different conditions in dayflower. This study will enhance the precision of the qRT-PCR quantification of candidate genes related to the adaptation significance of dayflower.

Rice Reference Genes (RRG): redefining reference genes in rice by mining RNA-seq datasets.

Reverse transcription quantitative real-time PCR (RT-qPCR) is esteemed for its precision and reliability, positioning it as the standard for evaluating gene expression. Selecting suitable reference genes is crucial for acquiring accurate data on target gene expression. However, identifying appropriate reference genes for specific rice tissues or growth conditions has been a challenge. To overcome this, we introduce the Rice Reference Genes (RRG) tool, which assists researchers in selecting reference genes for diverse experimental conditions in rice. This tool utilizes of 4,404 rice-derived RNA-seq datasets, categorized by five tissue types - leaf, root, seedling, panicle, and seed - and seven stress conditions (cold, disease, drought, heat, hormone, metal, and salt), along with corresponding control groups (mock). In this research, we employed the RRG web-based tool to identify candidate reference genes in rice leaves, roots, and seedlings exposed to salt and drought stress. These candidates were rigorously tested against conventionally established reference genes, confirming their accuracy and reliability. The RRG tool is designed to be user-friendly, allowing even those with limited experience to efficiently select optimal reference genes in rice with ease.

Reference gene selection for real-time qPCR in European flounder (Platichthys flesus) using organ-specific RNA-seq data

BackgroundThe European flounder is readily chosen as an experimental subject and model in physiological and ecotoxicological studies mostly because of its adaptability to laboratory conditions. Many studies utilise a quantitative PCR (qPCR) approach to ascertain the expression of target genes under experimental conditions. Such an approach relies heavily on the selection of reference genes with stable expression. Yet certain housekeeping genes are commonly used in this role, often without due consideration of their overall expression patterns. Therefore, new approaches should be developed to identify stable reference genes for a given species and to expand the general pool of genes suitable for the reference in qPCR analysis.Methods and resultsHere RNA-seq data of nine flounder organs led to identify four candidate genes of the most stable expression. It was achieved by differential expression analysis and tritoconstrictor script. Specific primers were designed for the complete ORF as well as for qPCR analysis. RT-qPCR efficiencies were tested on ORF amplicon templates. Most of the genes tested showed good amplification in a wide range of template dilutions (107-101), with a correlation coefficient (R2) ranging from 0.991 to 0.998 and a consistent efficiency (E) (Sybr Green I staining and TaqMan molecular probe).ConclusionsThe proposed approach based on differential expression analysis and a new bioinformatic tool is an appropriate selection method of candidates for reference genes in qPCR. The proposed approach, combining differential expression analysis with a new bioinformatics tool, provides an effective method for selecting reference gene candidates for qPCR. As a result, we can propose four genes (polr2f, yif1a, sf3b6, uba52), each with a set of validated primers, as suitable for consideration as reference genes in qPCR analysis in European flounder, an emerging model species.

Robust reference gene selection in Norway spruce: essential for real-time quantitative PCR across different tissue, stress and developmental conditions

Accurate gene expression analysis in Norway spruce (Picea abies) under diverse stress conditions requires the identification of stable reference genes for normalization. Notably, the literature lacks reports on suitable reference genes in Norway spruce. Here, we aimed to address this gap by identifying suitable reference genes for quantitative real-time PCR in Norway spruce across various stress conditions (drought, heat, pathogen infection) in seedlings, tissues (needle, phloem, root), and developmental stages (seedlings, mature trees). We evaluated the stability of 15 candidate reference genes and assessed their expression stability using five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder). Our results highlight ubiquitin-protein ligase (SP1), conserved oligomeric Golgi complex (COG7), and tubby-like F-box protein (TULP6) as the most stable reference genes, while succinate dehydrogenase (SDH5) and heat shock protein 90 (HSP90) were the least stable under various experimental conditions. COG7 and TULP6 are novel candidate reference genes reported for the first time. The expression stability of the identified reference genes was further validated using dehydrin-like protein 5 (PaDhn5) under drought conditions in Norway spruce. Pairwise variation analysis suggests that two reference genes were sufficient to normalize gene expression across all sample sets. This study provides a comprehensive analysis of reference gene stability under different experimental conditions and a catalog of genes for each condition, facilitating future functional genomic research in Norway spruce and related conifers.

Tbp and Hprt1 Are Appropriate Reference Genes for Splenic Neutrophils Isolated from Healthy or Tumor-Bearing Mice.

Background/Objectives: Neutrophils have recently gained significant attention due to their heterogeneity in tumor settings. The gene expression profiles of neutrophils from different tumor types are of great interest. Murine splenic neutrophils reflect the immune status of the organism and could be a source of tumor-associated neutrophils in tumor-bearing mice. However, information about appropriate reference genes for RT-qPCR analysis of murine neutrophils in the literature is lacking. The aim of this study was to identify stably expressed reference genes in murine splenic neutrophils. Methods: Bone marrow- and spleen-derived neutrophils were isolated from healthy C57Bl/6 and CBA/LacSto mice. Spleen-derived neutrophils were isolated from mice with Lewis lung carcinoma (LLC) and drug-resistant lymphosarcoma (RLS40). RNA was isolated and used for RT-qPCR analysis of 10 selected reference genes. Analysis of reference gene stability was performed using four different algorithms (BestKeeper, NormFinder, geNorm, ΔCt method), and comprehensive ranking was constructed using RefFinder. Results: The Ct values for the reference genes were in the range of 16.73-30.83 with the highest expression levels observed for B2m and the lowest for Sdha. Differences in the stability ranking performed by different algorithms were observed; however, the overall ranking of the studied reference genes was as follows, from most to least stably expressed: Tbp, Hprt1, Ywhaz, B2m, Gapdh, Actb, Sdha, Eef2, Rack1, and Rpl13a. Using Tbp or Rpl13a for RT-qPCR data normalization significantly affected the interpretation of target gene expression. Conclusions: Tbp and Hprt1 are recommended reference genes for murine splenic neutrophils regardless of their activation status.

Selection and validation of reference genes for quantitative expression analysis of regeneration-related genes in Cheilomenes sexmaculata by real-time qRT-PCR.

Regeneration is a fascinating phenomenon that has intrigued scientists for a long time. Cheilomenes sexmaculata (Fabricius), a zig-zag ladybird beetle, possesses a high capacity for limb regeneration. The molecular mechanics of the zig-zag ladybird beetle are under-explored. Current research trends are focused on uncovering functional genes associated with limb regeneration. Most of these investigations involve quantitative real-time PCR (qRT-PCR) for their rapid and accurate analysis of gene expression levels. Hence, a stable and suitable reference gene is required to normalize the gene expression data. In this study, five housekeeping genes were selected from the transcriptomics data (in-house unpublished data) of C. sexmaculata (Fabricius). The expression stabilities of the selected genes were evaluated under different time intervals post-amputation using geNorm, normFinder, and refFinder software. Actin was revealed to be the most stable housekeeping gene, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase. A target gene named engrailed (an important segment-forming gene) was used to validate the selected reference genes. The expression levels were found to be consistent with the transcriptomics results. According to our study, actin, along with elongation factor 2 and glyceraldehyde-3-phosphate dehydrogenase, serve as the most stable reference genes and are suitable for regeneration-related research. This study is a groundbreaking effort to identify the most stable reference gene for limb regeneration in C. sexmaculata (Fabricius), and the findings can be applied to other related insect species.

Reference Gene Selection for RT-qPCR Normalization in Toxoplasma gondii Exposed to Broxaldine.

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is widely used to accurately assess target gene expression. Evaluating gene expression requires the selection of appropriate reference genes. To identify reliable reference genes for Toxoplasma gondii (T. gondii) under varying concentrations of broxaldine (BRO), we employed the ΔCt method, BestKeeper, NormFinder, GeNorm, and the comprehensive web-based platform RefFinder to assess the expression stability of ten candidate reference genes in T. gondii. Herein, our findings reveal that the stability of these candidate reference genes is influenced by different experimental conditions. Under normal conditions, the most stable genes were TGME49_205470 and TGME49_226020. However, the most stable genes differed when BRO concentrations were at 1, 2, and 4 μg/mL. Across all samples, TGME49_247220 and TGME49_235930 were identified as the most stable reference genes. Moreover, we also confirmed the stability of TGME49_247220 and TGME49_235930 as reference genes through RT-qPCR assays. The present study provides a foundation for applying the RT-qPCR method to investigate target gene expression following BRO treatment in T. gondii.

Selection and validation of stable reference genes for gene expression analysis in response to sweetpotato weevil infestation (Cylas formicarius (Fabricius)) in [Ipomoea batatas (L.) Lam] and [Ipomoea mauritiana (Jacq.)

Selection and validation of stable reference genes for gene expression analysis in response to sweetpotato weevil infestation (Cylas formicarius (Fabricius)) in [Ipomoea batatas (L.) Lam] and [Ipomoea mauritiana (Jacq.)

Screening and stability verification of reference genes in Botrytis cinerea ZX2 fermentation

Botrytis cinerea, an airborne plant pathogen, holds the potential to synthesize sesquiterpenes, which have been used for the industrial production of abscisic acid. Previously, through our genetic technology, we obtained strain ZX2, whose main product 1´,4´-trans-ABA-diol is physiologically active in plants. In this study, 50 L of fed fermentation was carried out with ZX2 strain to study the stability of expression of TUA, TUB, ATC, EF-1, GAPDH, UCE and GTP genes. Four kinds of software (GeNorm, NormFinder, BestKeeper and Delta Ct) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by RefFinder. Based on the results, the ACT was the most stable gene. It was used to normalize the expression levels of two genes related to 1´,4´-trans-ABA-diol production (hmgr and bcaba3) when fed-batch fermentation. Guide the selection of appropriate internal reference genes during the fermentation process to accurately quantify the relative transcription levels of target genes in B.cinerea ZX2.

Selection of Reference Genes and HSP17.9A Expression Profiling in Heat-Stressed Grapevine Varieties

Background: “Touriga Franca” (TF) and “Touriga Nacional” (TN) are grapevine varieties cultivated in the ‘Douro Superior’ subregion (Northern Portugal) that experience stressful environmental conditions during the summer. Objectives: Aiming to profile the expression of stress-responsive genes by quantitative real-time PCR (qPCR) in TF and TN plants growing naturally, three candidate reference genes were first tested under controlled conditions. Methods: To simulate a summer’s day, TF and TN in vitro plants were exposed to 32 °C–3 h (heat acclimation) and 42 °C–1 h (severe heat stress, HS) followed by two recovery periods (32 °C–3 h and 24 °C–24 h). Leaf samples were collected at the end of each phase. Control plants were kept at 24 °C. Results: Among the candidate reference genes, the UBC and VAG pair showed the highest stability. The suitability of these genes for qPCR was validated by heat shock protein 17.9A (HSP17.9A) gene profiling. The HSP17.9A expression was up-regulated in both varieties and all experimental phases except in TF control plants. TN showed the highest HSP17.9A relative expression ratio after severe HS. Conclusions: TN responded faster than TF to the induced heat shocks. The UBC, VAG, and HSP17.9A genes revealed to be suitable for further qPCR assays in TF and TN grapevine varieties.

RGeasy: a reference gene analysis tool for gene expression studies via RT-qPCR

Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.

Transcriptome-guided selection of stable reference genes for expression analysis in spinach.

Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.

A systematic review on the selection of reference genes for gene expression studies in rodents: are the classics the best choice?

Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research-hamster, rat, and mouse-are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents.

Selection of Reference Genes of Flower Development in Ludisia discolor.

Background: RT-qPCR is a powerful strategy for recognizing the most appropriate reference genes, which can successfully minimize experimental mistakes through accurate normalization. Ludisia discolor, recognized for its ornamental value, features little, distinctive blossoms with twisted lips and gynostemium showing chiral asymmetry, together with striking blood-red fallen leaves periodically marked with golden blood vessels. Methods and Results: To ensure the accuracy of qRT-PCR, selecting appropriate reference genes for quantifying target gene expression levels is essential. This study aims to identify stable reference genes during the development of L. discolor. In this study, the entire floral buds, including the lips and gynostemium from different development stages, were taken as materials. Based upon the transcriptome information of L. discolor, nine housekeeping genes, ACT, HIS, EF1-α1, EF1-α2, PP2A, UBQ1, UBQ2, UBQ3, and TUB, were selected in this research study as prospect interior referral genes. The expression of these nine genes were found by RT-qPCR and afterwards comprehensively examined by four software options: geNorm, NormFinder, BestKeeper, and ΔCt. The outcomes of the analysis showed that ACT was the most steady gene, which could be the most effective inner referral gene for the expression evaluation of flower advancement in L. discolor. Conclusions: The results of this study will contribute to the molecular biology research of flower development in L. discolor and closely related species.

Brainstem transcriptomic changes in male Wistar rats after acute stress, comparing the use of duplex specific nuclease (DSN)

In this work, we have analyzed the transcriptomic changes in the brainstem of male Wistar rats 2 h after an acute stress exposure. We performed duplex-specific nuclease normalization of cDNA libraries and compared the results back-to-back for the first time. Based on our RNAseq data, we selected reference genes for RT-qPCR that are best suited for acute stress experiments. Most genes were upregulated. We detected a massive shift in neuropeptide Crh, Trh,Cga, Tshb, Uts2b, Tac4, Lep and neuropeptide receptor Hcrtr1, Sstr5, Bdkrb2, Crhr2 signaling, as well as glutamate Grin3b, Grm2 and GABA Gpr156, acetylcholine Chrm4,Chrne, adrenergic Adra2b receptors expression. A strong increase in the expression of intermediate filaments Krt83/Krt86/Krt80/Krt84/Krt87/Krt4/Krt76 and motor proteins Myo7a, Klc3 was detected. Remarkably, in the absence of astrocyte activation, we also observed signs of microglial activation at this time point. Both expression of anti-inflammatory cytokines Il13, Ccl24 and pro-inflammatory cytokine receptors Il9r, Il12rb1, Tnfrsf14, Tnfrsf13c, Tnfrsf25, Tnfrsf1b were increased. In the Wnt signaling pathway, we observed increased expression of ligands-receptors Wnt1, Wnt11, Ror2 and also negative regulators Notum, Sfrp5, Sost. RNAseq results after DSN treatment correlated at a high level with RNAseq results without DSN, but there was a proportion of genes that shifted their logFC values. They are mostly rare transcripts TPM 1–10 with higher 0.5–0.9 GC content.

Selecting reference genes for RT-qPCR studies involving the presence of B chromosomes in Psalidodon (Characiformes, Characidae).

B chromosomes are extra non-essential elements present in several eukaryotes. Unlike A chromosomes which are essential and present in all individuals of a species, B chromosomes are not necessary for normal functioning of an organism. Formerly regarded as genetically inactive, B chromosomes have been discovered to not only express their own genes, but also to exert influence on gene expression in A chromosomes. Recent studies have shown that, in some Psalidodon (Characiformes, Characidae) species, B chromosomes might be associated with phenotypic effects, such as changes in the reproductive cycle and gene expression. In this study, we aimed to establish stable reference genes for RT-qPCR experiments conducted on gonads of three fish species within Psalidodon genus, both in the presence and absence of B chromosomes. The stability of five selected reference genes was assessed using NormFinder, geNorm, BestKeeper, and RefFinder algorithms. We determined ppiaa and pgk1 as the most stable genes in P. fasciatus, whereas ppiaa and hmbsa showed the highest stability in P. bockmanni. For P. paranae, tbp and hprt1 were the most stable genes in females, and ppiaa and hprt1 were the most stable in males. We determined the most stable reference genes in gonads of three Psalidodon species considering the presence of B chromosomes. This is the first report of reference gene stability in the genus and provides valuable tools to better understand the effects of B chromosomes at gene expression level.

Selection of reference genes for expression profiling in biostimulation research of soybean

BackgroundPlant biostimulants constitute a promising environmentally friendly alternative for increasing crop yield and tolerance to unfavorable conditions. Among various types of such formulations, botanical extracts are gaining more recognition as products supporting plant performance. Moreover, novel tools such as cold-plasma or low-pressure microwave plasma discharge are being proposed as techniques that might improve their efficacy. Elucidation of the biostimulant’s mode of action requires complex research at a molecular level. Transcriptional changes occurring after biostimulant spraying might be investigated using RT-qPCR. However, this technique requires data normalization against stable endogenous controls.ResultsHere, we tested the expression stability of ten candidate genes in soybean plants exposed to various biostimulants treatment. Selection of the best-performing reference genes was conducted using four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). According to the obtained results, Bic-C2 (RNA-binding protein Bicaudal-C) and CYP (cyclophilin type peptidyl-prolyl cis–trans isomerase) showed highest expression stability, while expression of EF1B (elongation factor 1-beta) fluctuated the most among a tested set of candidate genes.ConclusionsOverall, we recommend using Bic-C2 together with CYP for the RT-qPCR data normalization in soybean biostimulation experiments. To our best knowledge, this is the first comprehensive study of reference genes stability in plants subjected to biostimulant treatment. The results of this study will aid in further biostimulant research in crop plants, facilitating analyses performed on the transcriptional level.Graphical

Selection of Reference Genes in Siraitia siamensis and Expression Patterns of Genes Involved in Mogrosides Biosynthesis.

Siraitia siamensis is a traditional Chinese medicinal herb. In this study, using S. siamensis cultivated in vitro, twelve candidate reference genes under various treatments were analyzed for their expression stability by using algorithms such as GeNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. The selected reference genes were then used to characterize the gene expression of cucurbitadienol synthase, which is a rate-limiting enzyme for mogroside biosynthesis. The results showed that CDC6 and NCBP2 expression was the most stable across all treatments and are the best reference genes under the tested conditions. Utilizing the validated reference genes, we analyzed the expression profiles of genes related to the synthesis pathway of mogroside in S. siamensis in response to a range of abiotic stresses. The findings of this study provide clear standards for gene expression normalization in Siraitia plants and exploring the rationale behind differential gene expression related to mogroside synthesis pathways.