Reference genes for quantitative real-time PCR normalization of Cenostigma pyramidale roots under salt stress and mycorrhizal association.
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ABSTRACT:
SUBMITTER: Frosi G
PROVIDER: S-EPMC8167929 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1 α ), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (C t ) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.
| S-EPMC4058273 | biostudies-literature
Project description:The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1?, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.
| S-EPMC3329553 | biostudies-literature
Project description:Despite its superiority for evaluating gene expression, real-time quantitative polymerase chain reaction (qPCR) results can be significantly biased by the use of inappropriate reference genes under different experimental conditions. Reaumuria soongorica is a dominant species of desert ecosystems in arid central Asia. Given the increasing interest in ecological engineering and potential genetic resources for arid agronomy, it is important to analyze gene function. However, systematic evaluation of stable reference genes should be performed prior to such analyses. In this study, the stabilities of 10 candidate reference genes were analyzed under 4 kinds of abiotic stresses (drought, salt, dark, and heat) within 4 accessions (HG010, HG020, XGG030, and XGG040) from 2 different habitats using 3 algorithms (geNorm, NormFinder, and BestKeeper). After validation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large unite (rbcL) expression pattern, our data suggested that histone H2A (H2A) and eukaryotic initiation factor 4A-2 (EIF4A2) were the most stable reference genes, cyclophilin (CYCL) was moderate, and elongation factor 1? (EF1?) was the worst choice. This first systematic analysis for stably expressed genes will facilitate future functional analyses and deep mining of genetic resources in R. soongorica and other species of the Reaumuria genus.
| S-EPMC4130609 | biostudies-literature
Project description:Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1α would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1α were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana.
| S-EPMC3534648 | biostudies-literature
Project description:Background:A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in quantitative real-time PCR (qRT-PCR) analyses. However, there is no absolute universality in reference genes among different species. It's hard to find an ideal reference gene to fit for different tissues and growth periods. Pitaya (Hylocereus) is commercially produced as a new fruit crop at a large scale in tropical and subtropical regions. To date, there is no report on the identification of the most reliable reference genes for qRT-PCR normalization in pitaya. Results:In this study, six candidate reference genes i.e. Actin(1), GAPDH, UBC(1), UBC(2) EF1-?(1) and histone(1) were selected from thirty-nine typical candidate reference genes to determine the most stable reference genes for qRT-PCR normalization in different tissues, temperature stresses and fruit developmental stages of pitaya. Among the six candidate reference genes, Actin(1) and EF1-?(1) were the most stable gene according to calculations of three statistical methods (GeNorm, NormFinder and BestKeeper) while UBC(1) and UBC(2) showed the lowest expression stability. The six candidate reference genes were further validated by comparing expression profiles of key genes related to betalain biosynthesis at flesh coloration stages of Guanhuahong (Hylocereus monacanthus) and Guanhuabai (H. undatus) pitayas. Actin(1) was recommended the best reference gene for accurate normalization of qRT-PCR data. Conclusions:In this study, the stability of the selected reference genes for normalizing the qRT-PCR data were identified from pitaya. Actin(1) was the most stably expressed genes in different tissues and fruit developmental stages in pitaya. The present work provides the first data of reference gene identification for pitaya and will facilitate further studies in molecular biology and gene function on Hylocereus and other closely related species.
| S-EPMC6613322 | biostudies-literature
Project description:Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression, Lilium, and particularly L. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely 18S rRNA, ACT, BHLH, CLA, CYP, EF1, GAPDH, SAND and TIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms, BHLH was superior to the other candidates when all the experimental treatments were analyzed together; CLA and EF1 were also recommended by two of the three algorithms. As for specific conditions, EF1 under various developmental stages, SAND under biotic stress, CYP/GAPDH under drought stress, and TIP41 under salinity stress were generally considered suitable. All the algorithms agreed on the stability of SAND and GAPDH under cold stress, while only CYP was selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level of LrLOX in leaves inoculated with B. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding of Lilium.
| S-EPMC4806604 | biostudies-literature
Project description:BACKGROUND:The use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis-halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted. RESULTS:Ten candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae. CONCLUSIONS:Overall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A. littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.
| S-EPMC4950632 | biostudies-literature
Project description:Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and geNorm suggested the optimal number of reference genes for normalization. RPL13a and RPL18 were the most suitable reference genes for developmental stages, tissues, and temperature treatments; RPL13a and RPS3 were the most suitable for pesticide and photoperiod treatments; RPS18 and RPL18 were the most suitable for the two sexes. We validated the normalized results using odorant-binding protein genes as target genes in different tissues. Compared with the selected suitable reference genes, the expression of OBP1 in antennae, abdomen, and wings, and OBP2 in antennae and wings were overestimated due to the instability of ACTb. These results identified several reliable reference genes in H. oblita for normalization, and are valuable for future molecular studies.
| S-EPMC7577503 | biostudies-literature
Project description:Melon (Cucumis melo. L) is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR), which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.
| S-EPMC3903635 | biostudies-literature
Project description:Haloxylon ammodendron plays an important role in maintaining the structure and function of the entire ecosystem where it grows. No suitable reference genes have been reported in H. ammodendron plants to date. In this study, a total of 8 reference genes (18S, ACT1, ACT7, UBC18, TUA5, GAPDH, EF-1? and UBQ10) were selected from the available trancriptome database, and the expression stability of these 8 candidate genes was validated under different abiotic stress with three different statistical algorithms (geNorm, NormFinder and BestKeeper). The results produced from different models were in agreement with each other essentially: 18S and TUA5 were the most stable genes under drought stress, 18S, the most stable gene under heat stress and mechanical damage, ACT7 and UBC18, stable under salt stress while TUA5 and GAPDH expressed constantly under mechanical damage, and ACT1 expressed steadily under cold conditions. Expression profiles of several stress response genes, including FT-5, FT-9, DREB2A and DREB2C, were further confirmed with various candidate reference genes. None of the candidate genes showed a constant expression among all tested samples. Hence, it's essential to use more than one reference gene in order to guarantee the accuracy of quantitative real-time PCR. The results of this study will contribute to the accuracy and reliability in transcripts quantification, which is of significance to transcription-based studies and applications in this important shrub H. ammodendron.
| S-EPMC5911265 | biostudies-literature