Selection Of Appropriate Reference Genes Research Articles

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.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Characterizing reference genes for high-fidelity gene expression analysis under different abiotic stresses and elicitor treatments in fenugreek leaves

BackgroundQuantifying gene expression is a critical aspect of applied genomics research across all organisms, and real-time PCR has emerged as a powerful tool for this purpose. However, selecting appropriate internal control genes for data normalization presents specific challenges. This study aimed to identify suitable reference genes for gene expression analysis under various conditions, encompassing salinity, low and high-temperature stresses, and different elicitor treatments. These treatments included titanium dioxide, cold plasma, 24-epibrassinolide, and melatonin, resulting in a total of 13 unique treatments and 148 treatment combinations applied to fenugreek plants.ResultsAs per the analysis performed with the BestKeeper tool, EEF-1α, and GAPDH were recognized as the most stable reference genes under the majority of conditions. Furthermore, the GeNorm and NormFinder tools identified β-tubulin and EEF-1α as the most stable reference genes. The findings of this research demonstrated that, although the stability of three reference genes expression was acceptable in almost all evaluated treatments, fluctuations in their expression were observed under the treatments of cold stress with TiO2 NPs application, cold plasma application with salinity stress, and cold plasma application with high-temperature stress compared to others. Simultaneously, the GeNorm analysis results demonstrated that in the mentioned treatments, relying on only one reference gene is inadequate. To corroborate the results, we examined the expression profile of the SSR gene, a pivotal gene in diosgenin biosynthesis, under all investigated treatments and treatment combinations. The outcomes suggested that employing stable reference genes yielded highly consistent results.ConclusionsThe varying expression patterns of the target genes emphasize the crucial need for precise optimization of experimental conditions and selecting stable reference genes to achieve accurate results in gene expression studies utilizing real-time PCR. These findings offer valuable insights into the selection of appropriate reference genes for gene expression analysis under diverse conditions using real-time PCR.

Selection and validation of reference genes for RT-qPCR normalization of porcine alveolar macrophages (PAMs) for PRRSV studies

Porcine alveolar macrophages (PAMs) are widely used for in vitro studies of porcine respiratory viruses. Gene expression in these cells is altered by viral infection and cellular immune response. Real-time reverse transcription polymerase chain reaction (RT-qPCR) is a powerful technique for analyzing these changes. In order to obtain reliable quantitative RT-qPCR data and come to sound conclusions, stable reference genes are needed for normalization of target gene expression. In the present study, we evaluated the expression stability of nine reference genes in PAMs during cultivation and upon porcine reproductive and respiratory syndrome virus (PRRSV) inoculation. Using geNorm and NormFinder algorithms, we identified PSAP and GAPDH as the most stable reference genes under all experimental conditions. The selected reference genes were used for the normalization of CD163 expression under different conditions. This study demonstrates that selection of appropriate reference genes is essential for normalization and validation of RT-qPCR data across all experimental conditions. This study provides a new set of stable reference genes for future studies with porcine respiratory viruses in PAMs.

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Megalurothrips usitatus (thysanoptera: thripidae).

Introduction: Gene expression analysis by reverse transcription quantitative polymerase chain reaction (qRT-PCR) has been widely used in research including insects. The selection of appropriate reference genes is the key to obtaining accurate and reliable results from qRT-PCR. However, studies on the expression stability of reference genes in Megalurothrips usitatus remain lacking. Methods: In this study, qRT-PCR was used to analyze the expression stability of candidate reference genes in M. usitatus. The expression levels of six candidate reference gene transcription of M. usitatus were analyzed. GeNorm, NormFinder, BestKeeper, and ΔCt were used to analyze the expression stability of M. usitatus treated with biological factors (developmental period treatment) and abiotic factors (light, temperature, insecticide treatment, respectively). Comprehensive stability ranking of candidate reference genes was recommended by RefFinder. Results and Discussion: Results showed that ribosomal protein S (RPS) was the most suitable expression in insecticide treatment. Ribosomal protein L (RPL) was the most suitable expression at developmental stage and light treatment, whereas elongation factor was the most suitable expression in temperature treatment. RefFinder was used to comprehensively analyze the above four treatments, and the results showed that RPL and actin (ACT) showed high stability in each treatment. Therefore, this study identified these two genes as reference genes in the qRT-PCR analysis of different treatment conditions of M. usitatus. Ourfindings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in M. usitatus.

Development of Reference Genes for Horticultural Plants

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is extensively applied technique to investigate the transcript abundance of target genes in various organisms. Selection of appropriate reference genes (RGs) for qRT-PCR normalization is a crucial prerequisite for accurately quantifying gene expression level. RGs should exhibit minimal variation in gene expression. However, the actual expression stability of RGs fluctuates greatly in different species or under different experimental conditions. Due to rapid advancements in next-generation sequencing (NGS) technology, it is no longer difficult to get massive transcriptome data, which has greatly promoted the development of RGs. In this review, we elaborate on the strategies for developing RGs using Northern blotting, expressed sequence tags (ESTs), qRT-PCR, and high-throughput technologies such as microarray and RNA-sequencing (RNA-Seq). The process for developing RGs based on RNA-Seq is further addressed, including processing and normalization of RNA-Seq data, evaluation of gene expression stability, and screening and validation of RGs. The most frequently used RGs in horticultural plants are summarized, and the strategies for developing these RGs are introduced in detail. The information provided here will help to design effective strategies for the development of RGs in horticultural plants, with a focus on using big data generated by RNA-Seq.

TBP, PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes’ expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.

Selection of appropriate reference genes in different tissues of Vaccinium dunalianum Wight by quantitative real-time PCR for gene expression studies

Quantitative real-time polymerase chain reaction (PCR) is a powerful tool for investigating relevant changes in gene expression. At present, there are no reports on reference genes for Vaccinium dunalianum Wight (V. dunalianum Wight). Here, the expression profiles of 15 frequently used reference genes were used to screen for appropriate reference genes in different tissues of V. dunalianum Wight collected in Yunnan and Wuding, China. The cycle threshold (Ct) values of genes in different samples were compared and analyzed. Furthermore, BestKeeper, NormFinder, and geNorm software were used to evaluate the stability of each candidate reference gene. Analysis using geNorm showed that the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A-2 (PP2A-2) were the most stable. In turn, the expression level of eukaryotic initiation factor-2 (EIF-4A-2) was the most stable as per analysis by NormFinder, while Bestkeeper revealed that actin-related protein-1 (ARP-1) was the most stably expressed. Comprehensive analysis and validation experiments showed that EIF-4A-2 and PP2A-2 were the most suitable reference genes for V. dunalianum Wight.

Pregnancy influences the selection of appropriate reference genes in mouse tissue: Determination of appropriate reference genes for quantitative reverse transcription PCR studies in tissues from the female mouse reproductive axis.

Selecting stably expressed reference genes which are not affected by physiological or pathophysiological conditions is crucial for reliable quantification in gene expression studies. This study examined the expression stability of a panel of twelve reference genes in tissues from the female mouse reproductive axis and the uterus. Gene expression studies were carried out using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). cDNA was synthesised from RNA extracted from hypothalami, pituitaries, ovaries and uteri of female mice at ages representing weaning, puberty and adulthood as well as pregnancy (13±1days post-coitus) (n=a minimum of 3 at each age and at pregnancy). The reference genes examined included 18s, Actb, Atp5b, B2m, Canx, Cyc1, Eif4a2, Gapdh, Rpl13a, Sdha, Ubc and Ywhaz. The RT-qPCR raw data were imported into the qBASE+ software to analyse the expression stability using GeNorm. These data were also subsequently analysed using other software packages (Delta CT, Normfinder, BestKeeper). A comprehensive ranking was conducted considering all stability rankings generated from the different software analyses. B2m and Eif4a2 deviated from the acceptable range for amplification efficiency and therefore were excluded from the further analyses. The stability of the reference genes is influenced by the software used for the analysis with BestKeeper providing markedly different results than the other analyses. GeNorm analysis of tissues taken at different ages but not including pregnant animals, indicated that the expression of the reference genes is tissue specific with the most stable genes being: in the hypothalamus, Canx and Actb; in the pituitary, Sdha and Cyc1; in the ovary, 18s, Sdha and Ubc; and in the uterus, Ywhaz, Cyc1, Atp5b, 18s and Rpl13a. The optimal number of reference genes to be used was determined to be 2 in the first three tissues while in the uterus, the V-score generated by the GeNorm analysis was higher than 0.15 suggesting that 3 or more genes should be used for normalisation. Inclusion of tissues from pregnant mice changed the reference genes identified as being the most stable: Ubc and Sdha were the most stable genes in the hypothalamus, pituitary and the ovary. The addition of pregnant tissue had no effect on the stability of the genes in uterus (Ywhaz, Cyc1, Atp5b, 18s and Rpl13a). Identification of these stable reference genes will be of use to those interested in studying female fertility and researchers should be alert to the effects of pregnancy on reference gene stability. This study also signifies the importance of re-examining reference gene stability if the experimental conditions are changed, as shown with the introduction of pregnancy as a new factor in this research.

Selection and validation of appropriate reference genes for RT-qPCR analysis of flowering stages and different genotypes of Iris germanica L

Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

Reference gene validation in Eotetranychus sexmaculatus (Acari: Tetranychidae) feeding on mite-susceptible and mite-resistant rubber tree germplasms.

Reliable reference genes are quite important in calculating gene transcript levels by using real-time quantitative reverse transcription-PCR (RT-qPCR). Eotetranychus sexmaculatus is known as a dangerous mite causing significant yield reduction of rubber tree latex; however, selection of appropriate reference genes for validation of target gene expression in E. sexmaculatus has not been conducted yet. In the present study, nine candidate reference genes were analyzed for their expression stability in different life stages of E. sexmaculatus by using common algorithms including comparative ΔCq method, geNorm, NormFinder and BestKeeper. In addition, a comprehensive analysis software (RefFinder) was used to assign an overall final rank for each candidate gene. The results showed that β-actin and β-TUB were the best two reference genes and were subjected to evaluate expression of two protective enzyme genes (EsCu/ZnSOD and EsCAT1) in E. sexmaculatus. We found that the expression of EsCu/ZnSOD and EsCAT1 in E. sexmaculatus feeding on mite-resistant rubber tree germplasm was significantly lower compared with those feeding on mite-susceptible germplasm. These results will facilitate research in revealing molecular mechanisms underlying rubber tree resistance to the spider mite.

Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress

BackgroundQuantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five ‘traditional’ RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database.ResultsExpression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition.ConclusionsOur results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.

Validation of Reference Genes for Quantitative Gene Expression Studies in Octopus minor

Widely distributed on tidal flats on the west and south coast of Korea, the long arm octopus Octopus minor is one of the important shellfish resources, as well as a key species in the siltymud tidal ecosystem. While several molecular studies have studied the genomes or the molecular phylogenetic relationship of O. minor with other cephalopod members, no studies to date have reported on gene expression in the long arm octopus. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a commonly used method in gene expression studies; however, the accuracy and reliability of the assay results depend strongly on selection of appropriate reference genes. In this study, we first evaluated stabilities of 8 reference genes (elongation factor 1-beta, elongation factor 2, ribosomal protein, TATA-binding protein-associated factor 172, histone H4, elongation factor-gamma, succinate-hydroxymethylglutarate CoA-transferase, and transferrin) of O. minor distributed in the different tissues using geNorm. Our results indicated that TF was the most stable of the selected reference genes from the various tissues. To illustrate the usefulness of the new reference genes, expression analysis of heat shock protein70 related to environmental stresses was undertaken. Accordingly, this study provides suitable reference genes for RT-qPCR analysis that will enable future studies of gene expression in O. minor and other related species in cephalopods in order to gain a better understanding of their molecular responses to environmental stresses.

Evaluation of the stability of standard reference genes of adipose-derived mesenchymal stem cells during in vitro proliferation and differentiation.

Mesenchymal stem cells (MSCs) from a variety of sources are being used in pre-clinical and clinical studies. The choice of optimal source for treatment of diseases requires quantitative evaluation of self-renewal, proliferation and differentiation potencies of MSCs. For this purpose, quantitative real-time polymerase chain reaction (qRT-PCR) technique is used to determine the expression of genes. qRT-PCR requires the normalization of the gene expression levels by the use of reference genes in order to obtain accurate and reliable results. There is a limited number of studies focused on the selection of reference genes that are appropriate and reliable for MSCs. Thus, no single reference gene has yet been found for use in the in vitro proliferation and differentiation of MSCs. The aim of this study is to investigate the stability of the expression of widely used reference genes during the in vitro proliferation and differentiation of human adipose-derived mesenchymal stem cells (hASCs). For this purpose, 13 reference genes commonly used in MSC studies were selected. As a result, the expression stabilities of EF1α, RPLP0 and RPL13A genes were found to be high and were predicted to be suitable for use as reference genes for normalization in hASC studies. The GAPDH was identified as the gene with the lowest expression stability and evaluated to be an unsuitable reference gene for hASC differentiation studies. This piece of information could be crucial for the selection of appropriate reference genes and accurate measurement of gene expression in hASC studies.

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

Reference Gene Selection for Quantitative Real-Time PCR Analyses of Acer palmatum under Abiotic Stress

Quantitative real-time reverse transcriptase PCR (qRT-PCR) technology has been extensively used to estimate gene expression levels, and the selection of appropriate reference genes for qRT-PCR analysis is critically important for obtaining authentic normalized data. Acer palmatum is an important colorful leaf ornamental tree species, and reference genes suitable for normalization of the qRT-PCR data obtained from this species have not been investigated. In this study, the expression stability of ten candidate reference genes, namely, Actin3, Actin6, Actin9, EF1α, PP2A, SAMDC, TIP41, TUBα, TUBβ and UBQ10, in two distinct tissues (leaves and roots) of A. palmatum under four different abiotic stress conditions (cold, heat, salt and drought) were investigated and assessed using three statistical methods (GeNorm, NormFinder and BestKeeper). The combinations of reference genes that showed stability in the different stressed samples differed. Specifically, Actin6, UBQ10 and Actin9 were the most stable reference genes in all the samples, and Actin3 and Actin6 wexre stably expressed in coldstressed leaves and roots. Actin3, Actin6 and UBQ10 were identified as an appropriate combination of reference genes for the analysis of heat-stressed leaves and roots, whereas the combination of Actin9, UBQ10 and Actin6 was deemed the most suitable for the analysis of salt-stressed leaves and roots. Similarly, Actin6 and UBQ10 exhibited stable expression in drought-stressed leaves and roots. Furthermore, the expression levels of CBF, Cu/Zn-SOD and HsfA1 were estimated to determine the reliability of the reference genes assessed in this study. This study revealed stable reference genes in A. palmatum that might be used for the normalization of qRT-PCR data obtained under various abiotic stresses.

Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions.

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Reference gene selection and validation by qRT-PCR during flower development and in different organs of Primula forbesii

ABSTRACTQuantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) is an effective and widely used method in gene expression studies. Selection of appropriate reference genes is key to generating reliable data using this technique and there are no published studies regarding the selection of reliable reference genes in the genus Primula. In this study, 10 candidate reference genes (28S, ACT, TUA, TUB, HIS, GAPDH, RPL18a, STPP, CSD and RNA pol II), were chosen from the transcriptome database in Primula forbesii Franch., and their expression levels were assessed by qRT-PCR in different flower developmental stages and different organs. Four software programs; geNorm, NormFinder, Bestkeeper, and Ref-Finder were used to validate the stability and suitability of potential reference genes. Comprehensive results showed that GAPDH was the most stable reference gene for P. forbesii, while TUA and TUB were the most unstable ones in flower development and different organs, respectively. Finally, to illustrate the usefulness of the top-ranked reference genes, the expression profile of the P. forbesii linalool synthase/nerolidol synthase gene (PfLIS/NES) was analysed, which highlighted the importance of proper reference gene selection. This work represents the first systematic study of reference gene stability in Primula and lays the foundation for future research into Primula gene expression patterns.

Selection of appropriate reference genes for RT-qPCR analysis under abiotic stress and hormone treatment in celery.

Celery is one of the most important vegetable crop and its yield and quality is influenced by many environmental factors. Researches on gene expression not only help to unravel the molecular regulatory mechanism but also identify the key genes in the biological response. RT-qPCR is a commonly used technology to quantify the gene expression. Selecting an appropriate reference gene is an effective approach to improve the accuracy of RT-qPCR assay. To our knowledge, the evaluation of reference genes under different treatments in celery has not been reported yet. In this study, the expression stabilities of eight candidate reference genes (ACTIN, eIF-4α, GAPDH, TBP, TUB-A, UBC, TUB-B, and EF-1α) under abiotic stresses (heat, cold, drought, and salt) and hormone treatments (SA, MeJA, GA, and ABA) were detected. The expression stabilities of candidate genes were compared and ranked by geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder programs. The results calculated by different programs were not completely consistent. Considering the comprehensive analysis results, ACTIN was the most stable reference gene and TUB-B showed the worst expression stabilities under the selected abiotic stress and hormone treatments in celery. The reliability of reference genes was further confirmed by the normalization of CAT1 gene under drought stress. This study presented evidences and basis to select the appropriate reference genes under different treatments in celery.