Selection Of Stable Reference Genes Research Articles

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.

Selection of Stable Reference Genes for QRT-PCR in Tree Peony 'Doulv' and Functional Analysis of PsCUC3.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (β-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

Validation of reference gene stability for normalization of RT-qPCR in Phytophthora capsici Leonian during its interaction with Piper nigrum L.

The selection of stable reference genes for the normalization of reverse transcription quantitative real-time PCR (RT-qPCR) is generally overlooked despite being the crucial element in determining the accuracy of the relative expression of genes. In the present study, the stability of seven candidate reference genes: actin (act), α-tubulin (atub), β-tubulin (btub), translation elongation factor 1-α (ef1), elongation factor 2 (ef2), ubiquitin-conjugating enzyme (ubc) and 40S ribosomal protein S3A (ws21) in Phytophthora capsici has been validated. The validation was performed at six infection time points during its interaction with its susceptible host Piper nigrum, two developmental stages, and for the combined dataset. Four algorithms: geNorm, NormFinder, BestKeeper, and the ΔCt method were compared, and a comprehensive ranking order was produced using RefFinder. The overall analysis revealed that ef1, ws21, and ubc were identified as the three most stable genes in the combined dataset, ef1, ws21, and act were the most stable at the infection stages, and, ef1, btub, and ubc were most stable during the developmental stages. These findings were further corroborated by validating the P. capsici pathogenesis gene NPP1 expression. The findings are significant as this is the first study addressing the stability of reference genes for P. capsici–P. nigrum interaction studies.

Selection of stable reference genes for qPCR expression of Colletotrichum lindemuthianum, the bean anthracnose pathogen

Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat”. However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), β-tub (β-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and β-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Herbaceous Peony (Paeonia lactiflora Pall.) in Response to Drought Stress

Herbaceous peony (Paeonia lactiflora Pall.), as a high-end cut flower in the international market, has high ornamental and medicinal values. But in Northern China, drought is a major environmental factor influencing the growth and development of P. lactiflora. Quantitative real-time polymerase chain reaction (qRT-PCR) can evaluate gene expression levels under different stress conditions, and stable internal reference is the key for qRT-PCR. At present, there is no systematic screening of internal reference for correcting gene expressions of P. lactiflora in response to drought stress. In this study, 10 candidate genes [ubiquitin (UBQ2), UBQ1, elongation factor 1-α (EF-1α), Histidine (His), eukaryotic initiation factor (eIF), tubulin (TUB), actin (ACT), UBQ3, ACT2, RNA polymerase II (RNA Pol II)] were chosen, and 4 analysis methods were used to compare the stabilities for these 10 genes coping with drought stress. Due to the difference of operation methods, the results of different analysis were distinct, and the final comprehensive analysis indicated that EF-1α was a relatively stable internal reference gene for P. lactiflora under drought stress. Also, UBQ1 and UBQ2 were the best reference gene combination according to GeNorm analysis. This study will lay a foundation for screening the key genes of P. lactiflora in response to drought stress.

Assessment of Suitable Reference Genes for qRT-PCR Normalization in Eocanthecona furcellata (Wolff).

Simple SummaryEocanthecona furcellata (Wolff) is an important polyphagous predatory natural enemy insect for agriculture and forestry production. In this paper, we screened nine commonly used reference genes β-1-TUB, RPL4, RPL32, RPS17, RPS25, SDHA, GAPDH2, EF2, and UBQ. Five methods, Ct value, geNorm, NormFinder, BestKeeper, and RefFinder, were used to assess the stability of gene expression at different developmental stages, in different tissues of male and female adults, under different temperatures and starvation treatments. Finally, stable reference genes were screened under different experimental conditions, which laid the foundation for further study of E. furcellata gene function.Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) is a widely used tool for measuring gene expression; however, its accuracy relies on normalizing the data to one or more stable reference genes. Eocanthecona furcellata (Wolff) is a polyphagous predatory natural enemy insect that preferentially feeds on more than 40 types of agricultural and forestry pests, such as those belonging to the orders Lepidoptera, Coleoptera, and Hymenoptera. However, to our knowledge, the selection of stable reference genes has not been reported in detail thus far. In this study, nine E. furcellata candidate reference genes (β-1-TUB, RPL4, RPL32, RPS17, RPS25, SDHA, GAPDH2, EF2, and UBQ) were selected based on transcriptome sequencing results. The expression of these genes in various samples was examined at different developmental stages, in the tissues of male and female adults, and after temperature and starvation treatments. Five algorithms were used, including ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder, to evaluate reference gene expression stability. The results revealed that the most stable reference genes were RPL32 and RPS25 at different developmental stages; RPS17, RPL4, and EF2 for female adult tissue samples; RPS17 and RPL32 for male adult tissue samples; RPS17 and RPL32 for various temperature treatments of nymphs; RPS17 and RPS25 for nymph samples under starvation stress; and RPS17 and RPL32 for all samples. Overall, we obtained a stable expression of reference genes under different conditions in E. furcellata, which provides a basis for future molecular studies on this organism.

Selection of stable reference genes for RT–qPCR in Salmo trutta

To identify suitable reference genes for Salmo trutta, real-time fluorescent quantitative PCR (RT–qPCR) technology was used to determine the expression of eight candidate genes in 11 tissues (heart, liver, spleen, head kidney, muscle, brain, gills, ovary, intestine, skin, testis) of S. trutta. Concurrently, expression of these eight reference genes was assessed in 1 d and 30 d S. trutta embryos and muscle tissue of 90 d and 180 d S. trutta. Finally, the eight candidate genes were comprehensively screened using four types of screening software (GeNorm, NormFinder, BestKeeper and RefFinder), and the stability of their expression in different tissues, in response to bacterial stress and during different developmental stages in S. trutta tissues was ranked. The results indicated that the eight candidate genes were specifically amplified in each tissue of S. trutta, with band sizes ranging from 73 bp to 150 bp. The stability ranking of the eight candidate genes in different tissues of healthy adult S. trutta was hprt1>β-actin>EF1a>RPL13a>RPS29>β2M>GAPDH>28 S rRNA. The ranking of the stability of expression in different tissues of S. trutta artificially infected with Aeromonas salmonicida was RPS29>hprt1>β2M>RPL13a>EF1a>β-actin>28 S rRNA>GAPDH. The ranking of the stability of expression in the tissues of S. trutta at different developmental stages was RPS29>GAPDH>hprt1>RPL13a>EF1a>28 S rRNA>β-actin>β2M. Comprehensive analysis revealed that expression of the hprt1 gene was relatively stable under different conditions. These results could guide the quantitative analysis of functional gene expression in S. trutta and calibrate the expression of major functional genes of S. trutta, facilitating the acquisition of more accurate, reliable and meaningful results.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Paeonia ostii T. Hong et J. X. Zhang Leaves Exposed to Different Drought Stress Conditions

The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy. Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors. In this study, the expression profiles of ten candidate genes, ACT1, ACT2, GAPDH, eIF1, eIF2, α-TUB, β-TUB, TBP, RNA Pol II and RP II, were calculated for a suitable reference gene selection in Paeonia ostii T. Hong et J. X. Zhang leaves under various drought stress conditions. Data were processed by the four regularly used evaluation software. A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one. In addition, the geNorm program provided the optimal choice of two reference gene combination, RNA Pol II and β-TUB, for qRT-PCR normalization in P. ostii subjected to different drought stress levels. Our research provided convenience for gene expression analysis in P. ostii under drought stress and promoted research of effective methods to alleviate P. ostii drought stress in the future.

Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits.

The selection and validation of stably expressed reference genes is key for accurately quantifying the mRNA abundance of genes under different treatments. In the rabbit model of fasting caecotrophy, reports about the selection of stable reference genes are not available. This study aims to screen suitable reference genes in different tissues (including uterus, cecum, and liver) of rabbits between control and fasting caecotrophy groups. RT-qPCR was used to analyze the expression levels of eight commonly used reference genes (including GAPDH, 18S rRNA, B2M, CYP, HPRT1, β-actin, H2afz, Ywhaz), and RefFinder (including geNorm, NormFinder, and BestKeeper) was used to analyze the expression stability of these reference genes. Our results showed that the most stable reference genes were different in different tissues and treatments. In the control and fasting caecotrophy groups, CYP, GAPDH and HPRT1 were proven to be the top stable reference genes in the uterus, cecum, and liver tissues, respectively. GAPDH and Ywhaz were proven to be the top two stable reference genes among uterus, cecum, and liver in both control and fasting caecotrophy groups. Our results indicated that the combined analysis of three or more reference genes (GAPDH, HPRT1, and Ywhaz) are recommended to be used for RT-qPCR normalization in the rabbit model of fasting caecotrophy, and that GAPDH is a better choice than the other reference genes for normalizing the relative expression of target genes in different tissues of fasting caecotrophy rabbits.

Stability evaluation of candidate reference genes for RT-qPCR normalization in Lasioderma serricorne (F.)

Lasioderma serricorne (Fabricius) is an important insect pest of stored products worldwide. The real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used to analyze gene expression across various biological processes. For accurate gene expression analyses using RT-qPCR, selection of stable reference genes to normalize RT-qPCR data is a prerequisite. However, the lack of studies on validation of reference genes in L. serricorne limits application of RT-qPCR in L. serricorne. In this study, the expression stability of eleven candidate reference genes (Actin, ARF1, β-Tubulin, EF1α, SYN6, TBP1, RPL3, RPL12, RPL13a, RPL32, 18S rRNA using five algorithms (geNorm, NormFinder, BestKeeper, ΔCt and RefFinder) in L. serricorne under different experimental and biotic conditions was assessed. The results showed that the best combinations of reference genes that could be used as internal controls in L. serricorne were 18S rRNA and RPL13a for different development stages; 18S rRNA and RPL3 for different larval tissues; RPL3 and RPL13a for different temperatures; RPL32, RPL12 and Actin for starvation. These results provided for the first time a comprehensive list of stable reference genes for the normalization of RT-qPCR analyses in L. serricorne and will benefit the in-depth molecular biology research in the future.

Selection of q RT-PCR reference genes for Amomum tsaoko seeds during dormancy release

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.

Selection of stable reference genes for real-time quantitative PCR in honey bee pesticide toxicity studies

Honey bees are exposed to various pesticides via both acute and chronic routes. Transcriptional profiling based on quantitative real-time PCR could be used as a representative method to assess the effects of pesticides on honey bee physiology. In this method, the identification of reference genes left unaffected by pesticide treatment is required for accurate analysis; however, there have been no reports regarding such reference genes. In this study, the expression level fluctuations of two putative reference genes (arf1 and rab1), along with three commonly used reference genes (rpS18, actin, and gapdh), were investigated in different stages and tissues following treatment with fluvalinate, coumaphos, imidacloprid, and flupyradifurone, and their performances were compared using geNorm, BestKeeper, NormFinder and RefFinder. The expression stability of rab1a was the highest, followed by that of arf1, rpS18, actin, and gapdh, suggesting that rab1 and arf1 can be employed as reliable reference genes in pesticide studies.

Selection and Validation of Reference Genes for Gene Expression Analysis in Tuta absoluta Meyrick (Lepidoptera: Gelechiidae).

Simple SummaryReference genes are critical for standardizing expression data of RT-qPCR across samples of organisms under different experimental conditions. However, most commonly used reference genes may not be stably expressed leading to a risk of misinterpretation of results. In our study, nine reference genes were evaluated in Tuta absoluta (a destructive pest of tomato) at different developmental stages, tissues, 20-hydroxyecdysone (20E) and insecticide treatments. Finally, the expression profile of indicator gene EcR after 20E treatment was evaluated to verify the accuracy of the results. This study is essential for improving accuracy and reliability to normalize gene expression data in T. absoluta and provides a useful strategy for other insects.The tomato leaf miner, Tuta absoluta is a destructive pest of tomato. The leaf-mining activities of its larvae can cause significant yield losses. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used to measure gene expression, and the selection of stable reference genes for calibration and standardization is critical for accurate use of RT-qPCR. We studied the stable expression of nine common housekeeping genes in T. absoluta. These were examined at different developmental stages, in larval tissues, as well as those induced by exposure to 20E and insecticides. Four dedicated algorithms (geNorm, BestKeeper, NormFinder, and ΔCt method) and online tool (RefFinder) were used to analyze and rank the tested reference genes. Based on the standardized gene expression data of target gene ecdysone receptor (EcR), the applicability of specific reference genes was verified. The results clarify that the optimal internal reference genes vary greatly under different experimental conditions. GAPDH and RPS11 were the best reference genes for developmental stages; RPL28 and RPL10 for different tissues; EF1α and RPL28 for 20E treatment; EF1α and RPL7A for insecticide treatments. The most suitable reference genes in all experimental conditions are EF1α and RPL28.

Reference Gene Selection for qRT-PCR Normalization in Iris germanica L.

Quantitative real-time PCR (qPCR) is an effective and widely used method to analyze expression patterns of target genes. Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR. In Iris germanica L., no studies have yet been published regarding the evaluation of potential reference genes. In this study, nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder). The results revealed that ACT11 (Actin 11) and EF1α (Elongation factor 1 alpha) were the most stable reference genes in different tissues, whereas TUA (Tubulin alpha) and UBC9 (Ubiquitin-protein ligase 9) were the most stable ones in different flower developmental stages. UBC9 and ACT11 were the most stable reference genes in all of the tested samples, while the SAMDC (S-Adenosylmethionine decarboxylase) showed the least stability. Finally, to validate the suitability of the selected reference genes, the relative expression level of IgTPS (beta-caryophyllene synthase) was assessed and highlighted the importance of suitable reference gene selection. This work constitutes the first systematic evaluation of potential reference genes in I. germanica and provides guidelines for future research on gene function and molecular mechanisms on I. germanica and related species.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses.

White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

Statistical differences resulting from selection of stable reference genes after hypoxia and hypothermia in the neonatal rat brain.

Real-time reverse transcription PCR (qPCR) normalized to an internal reference gene (RG), is a frequently used method for quantifying gene expression changes in neuroscience. Although RG expression is assumed to be constant independent of physiological or experimental conditions, several studies have shown that commonly used RGs are not expressed stably. The use of unstable RGs has a profound effect on the conclusions drawn from studies on gene expression, and almost universally results in spurious estimation of target gene expression. Approaches aimed at selecting and validating RGs often make use of different statistical methods, which may lead to conflicting results. Based on published RG validation studies involving hypoxia the present study evaluates the expression of 5 candidate RGs (Actb, Pgk1, Sdha, Gapdh, Rnu6b) as a function of hypoxia exposure and hypothermic treatment in the neonatal rat cerebral cortex–in order to identify RGs that are stably expressed under these experimental conditions–using several statistical approaches that have been proposed to validate RGs. In doing so, we first analyzed RG ranking stability proposed by several widely used statistical methods and related tools, i.e. the Coefficient of Variation (CV) analysis, GeNorm, NormFinder, BestKeeper, and the ΔCt method. Using the Geometric mean rank, Pgk1 was identified as the most stable gene. Subsequently, we compared RG expression patterns between the various experimental groups. We found that these statistical methods, next to producing different rankings per se, all ranked RGs displaying significant differences in expression levels between groups as the most stable RG. As a consequence, when assessing the impact of RG selection on target gene expression quantification, substantial differences in target gene expression profiles were observed. Altogether, by assessing mRNA expression profiles within the neonatal rat brain cortex in hypoxia and hypothermia as a showcase, this study underlines the importance of further validating RGs for each individual experimental paradigm, considering the limitations of the statistical methods used for this aim.

Selection and validation of reference genes for normalisation of gene expression in ischaemic and toxicological studies in kidney disease.

Normalisation to standard reference gene(s) is essential for quantitative real-time polymerase chain reaction (RT-qPCR) to obtain reproducible and comparable results of a gene of interest (GOI) between subjects and under varying experimental conditions. There is limited evidence to support selection of the commonly used reference genes in rat ischaemic and toxicological kidney models. Employing these models, we determined the most stable reference genes by comparing 4 standard methods (NormFinder, qBase+, BestKeeper and comparative ΔCq) and developed a new 3-way linear mixed-effects model for evaluation of reference gene stability. This new technique utilises the intra-class correlation coefficient as the stability measure for multiple continuous and categorical covariates when determining the optimum normalisation factor. The model also determines confidence intervals for each candidate normalisation gene to facilitate selection and allow sample size calculation for designing experiments to identify reference genes. Of the 10 candidate reference genes tested, the geometric mean of polyadenylate-binding nuclear protein 1 (PABPN1) and beta-actin (ACTB) was the most stable reference combination. In contrast, commonly used ribosomal 18S and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were the most unstable. We compared the use of PABPN1×ACTB and 2 commonly used genes 18S and GAPDH on the expression of 4 genes of interest know to vary after renal injury and expressed by different kidney cell types (KIM-1, HIF1α, TGFβ1 and PECAM1). The less stable reference genes gave varying patterns of GOI expression in contrast to the use of the least unstable reference PABPN1×ACTB combination; this improved detection of differences in gene expression between experimental groups. Reduced within-group variation of the now more accurately normalised GOI may allow for reduced experimental group size particularly for comparison between various models. This objective selection of stable reference genes increased the reliability of comparisons within and between experimental groups.

Genome wide search to identify reference genes candidates for gene expression analysis in Gossypium hirsutum

BackgroundCotton is one of the most important commercial crops as the source of natural fiber, oil and fodder. To protect it from harmful pest populations number of newer transgenic lines have been developed. For quick expression checks in successful agriculture qPCR (quantitative polymerase chain reaction) have become extremely popular. The selection of appropriate reference genes plays a critical role in the outcome of such experiments as the method quantifies expression of the target gene in comparison with the reference. Traditionally most commonly used reference genes are the “house-keeping genes”, involved in basic cellular processes. However, expression levels of such genes often vary in response to experimental conditions, forcing the researchers to validate the reference genes for every experimental platform. This study presents a data science driven unbiased genome-wide search for the selection of reference genes by assessing variation of > 50,000 genes in a publicly available RNA-seq dataset of cotton species Gossypium hirsutum.ResultFive genes (TMN5, TBL6, UTR5B, AT1g65240 and CYP76B6) identified by data-science driven analysis, along with two commonly used reference genes found in literature (PP2A1 and UBQ14) were taken through qPCR in a set of 33 experimental samples consisting of different tissues (leaves, square, stem and root), different stages of leaf (young and mature) and square development (small, medium and large) in both transgenic and non-transgenic plants. Expression stability of the genes was evaluated using four algorithms - geNorm, BestKeeper, NormFinder and RefFinder.ConclusionBased on the results we recommend the usage of TMN5 and TBL6 as the optimal candidate reference genes in qPCR experiments with normal and transgenic cotton plant tissues. AT1g65240 and PP2A1 can also be used if expression study includes squares. This study, for the first time successfully displays a data science driven genome-wide search method followed by experimental validation as a method of choice for selection of stable reference genes over the selection based on function alone.

Reference Gene Expression in Adipose-Derived Stromal Cells Undergoing Adipogenic Differentiation.

Adipose-derived stromal cells (ASCs) are becoming increasingly attractive as cellular therapy products. Their differentiation potential, the secretion of growth and differentiation factors, and the ability to cryopreserve the cells over extended periods are important features. Changes in experimental conditions result in changes in gene expression, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an important tool for measuring these changes. There is, however, the potential to introduce technical bias in the process, which can be diminished through the selection of stable reference genes (RGs). Using geNorm software, in this in vitro study we explore the effects that adipogenic differentiation for a 21 day induction period, cryopreservation (freshly isolated ASCs or previously cryopreserved/frozen ASCs), and culture medium supplementation (fetal bovine serum vs. pooled human platelet lysate) have on the stability of 11 RGs. We found that RG stability is markedly affected by the different experimental conditions. Of the RGs assessed, YWHAZ, HPRT, TBP, and ACTB were stably expressed genes under all experimental conditions. We recommend that a panel of stable RGs should be selected before studying gene expression during adipogenesis, and that this is based on the experimental condition(s) being investigated.Impact StatementAs the use of adipose-derived stromal cells (ASCs) in clinical trials increases, so does the amount of experimental data from research groups, many of which use human ASCs to study adipogenesis in obesity. Different conditions are constantly being applied to ASCs in vitro, to obtain a therapeutic product for potential downstream applications. Few articles have looked at the effect of different conditions on ASC reference gene (RG) expression and stability, which was the aim of this research, as such this article will assist other researchers to make an informed decision about RG selection for gene expression studies using ASCs including those for adipogenesis.

Reference genes selection for Calotropis procera under different salt stress conditions.

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.