Reference Genes For Expression Normalization Research Articles

Selection of Reference Genes for Expression Normalization by RT-qPCR in Dracocephalum moldavica L.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Selection of the Reference Gene for Expression Normalization in Salsola ferganica under Abiotic Stress.

Salsola ferganica is a natural desert herbaceous plant in the arid area of western and northwestern China. Because of its salt tolerance and drought resistance, it is of great significance in desert afforestation and sand-fixing capacity. There has been much research on the genes involved in plants under desert stresses in recent years. The application of the best internal reference genes for standardization was a critical procedure in analyzing the gene expression under different types. Even so, the reference gene has not been reported in the application of gene expression normalization of S. ferganica. In this study, nine reference genes (TUA-1726, TUA-1760, TUB, GAPDH, ACT, 50S, HSC70, APT, and U-box) in S. ferganica were adopted and analyzed under six different treatments (ABA, heat, cold, NaCl, methyl viologen (MV), and PEG). The applicability of candidate genes was evaluated by statistical software, including geNorm, NormFinder, BestKeeper, and RefFinder, based on their stability values in all the treatments. These results indicated that the simultaneous selection of two stable reference genes would fully standardize the optimization of the normalization research. To verify the feasibility of the above internal reference genes, the CT values of AP2/ERF transcription factor family genes were standardized using the most (ACT) and least (GAPDH) stable reference genes in S. ferganica seedlings under six abiotic stresses. The research showed that HSC70 and U-box were the most appropriate reference genes in ABA stressed samples, and ACT and U-box genes were the optimal references for heat-stressed samples. TUA-1726 and U-box showed the smallest value in gene expression levels of cold treatment. The internal reference groups of the best applicability for the other samples were U-box and ACT under NaCl treatment, ACT and TUA-1726 under MV stress, HSC70 and TUB under PEG treatment, and ACT in all samples. ACT and U-box showed higher stability than the other genes based on the comprehensive stability ranking of RefFinder, as determined by the geometric mean in this study. These results will contribute to later gene expression studies in other closely related species and provide an important foundation for gene expression analysis in S. ferganica.

Selection and Verification of Appropriate Reference Genes for Expression Normalization in Cryptomeria fortunei under Abiotic Stress and Hormone Treatments.

Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification.

Reference genes for the study of herbicide stress responses in Leptochloa chinensis (L.) Nees and estimation of ACCase expression in cyhalofop-butyl resistant populations

Cyhalofop-butyl resistance in Leptochloa chinensis (L.) Nees is a threat to rice production. Qualitative changes to the acetyl-CoA carboxylase gene (ACCase) have been reported to induce cyhalofop-butyl resistance in some weed species, but the role of ACCase in cyhalofop-butyl resistance through quantitative changes remains uncertain. The accurate assessment of transcriptional changes in the functional genes associated with herbicide resistance in L. chinensis is challenging owing to the lack of available reference genes for expression normalization. Here, we selected nine candidate reference genes in L. chinensis and assessed their transcription stability in populations susceptible and resistant to cyhalofop-butyl. Transcription stability was compared under conditions of herbicide stress and control conditions using BestKeeper, NormFinder, and geNorm. Elongation factor 1 alpha, eukaryotic initiation factor 4A, and cap-binding protein CBP20 were the most stable reference genes under cyhalofop-butyl treatment. Transcription levels of ACCase were evaluated in seven resistant populations, one of which showed higher transcription than the susceptible population after 24 h cyhalofop-butyl treatment. However, the slight up-regulation of ACCase (approximately 2.0-fold) is unlikely to be responsible for the high resistance levels in these populations of L. chinensis.

Transcriptome-based validation of proper reference genes for reverse trascription quantitative PCR analysis of Sinocalycanthus chinensis

Reverse transcription quantitative PCR is a widely used method to detect gene expressions. To obtain accurate expression results, the selection of proper reference genes is important and necessary. However, related works concerning reference gene selection have not been carried on many plant species, especially endangered ones. The aim of the present study was to select dependable reference genes for expression normalization of an endangered plant species with medicinal and ornamental value: Sinocalycanthus chinensis (Calycanthaceae). Nine reference genes with stable expressions were chosen for further analysis according to transcriptomic sequencing data from S. chinensis. The expression stability of these candidate genes in inner and outer petals at different floral developmental stages and in many different tissues was then analyzed with the geNorm and NormFinder software. The reference genes were evaluated by normalizing the expression of the anthocyanidin synthase gene in the outer and inner petals at different floral developmental stages to further verify the expression stability of these genes. Elongation factor 1-alpha (ScEF) and 50S ribosomal protein L27 were found to be the two most stable genes in the overall ranking of all the samples and different tissues. Furthermore, ScEF and protein phosphatase 2A were stably expressed in all petal samples. Moreover, among the nine candidate reference genes, phosphoglycerate kinase performed poorly in all sample sets. Our results will help to obtain reliable expression data in molecular studies of S. chinensis.

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment.

Papaver somniferum L. is an important medical plant that produces analgesic drugs used for the pain caused by cancers and surgeries. Recent studies have focused on the expression genes involved in analgesic drugs biosynthesis, and the real-time quantitative polymerase chain reaction (RT-qPCR) technique is the main strategy. However, no reference genes have been reported for gene expression normalization in P. somniferum. Herein, nine reference genes (actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin 2 (CYP2), elongation factor 1-alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic (GAPC2), nuclear cap-binding protein subunit 2 (NCBP2), protein phosphatase 2A (PP2A), TIP41-like protein (TIP41), and tubulin beta chain (TUB)) of P. somniferum were selected and analyzed under five different treatments (cold, drought, salt, heavy metal, and hormone stress). Then, BestKeeper, NormFinder, geNorm, and RefFinder were employed to analyze their gene expression stability. The results reveal that NCBP2 is the most stable reference gene under various experimental conditions. The work described here is the first report regarding on reference gene selection in P. somniferum, which could be used for the accurate normalization of the gene expression involved in analgesic drug biosynthesis.

Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs.

Baphicacanthus cusia (Nees) Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV) irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid), and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.

Reference genes for RT-qPCR analysis in Citrus and Poncirus infected by zoospores of Phytophthora parasitica

Phytophthora species are highly destructive phytopathogens, associated with massive damage in natural ecosystems and agriculture. Citrus production is also affected, mainly by the hemibiothrophic oomycete Phytophthora parasitica, which causes root rot and gummosis. Poncirus trifoliata and Citrus sunki (two rootstocks widely used in citrus orchards) pose a resistance and a susceptible interaction with P. parasitica, respectively, which makes them suitable models to study plant defense mechanisms. Gene expression analysis is a very important tool in this type of study, in particular PCR (RT-qPCR). Hence, it is crucial to use appropriate reference genes for expression normalization. Our aim was to evaluate the stability of several candidate reference genes to determine which set is best suited for normalization in citrus infected with P. parasitica. We evaluated five candidate reference genes selected from the database CitEST. GeNorm and NormFinder algorithms were used to assess the best reference genes. We found that the more stable genes to be used for RT-qPCR analysis in P. trifoliata plants were GAPC2 and F-BOX, while EGIDH and GAPC2 were more suitable to C. sunki. These four genes were found to be excellent normalizers, being stable throughout the infection regardless of pathogen attack or symptom development.

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.

Reference genes selection for transcriptional profiling in blood of HD patients and R6/2 mice.

Huntington's disease is a neurodegenerative disorder characterized by transcriptional alterations both in central and peripheral tissues. Therefore, the identification of a transcriptional signature in an accessible tissue can meaningfully complement current efforts in clinical biomarker development. Gene expression normalization represents an essential step in transcriptional signatures identification, and since many reference genes show altered expressions in several pathologies, the definition of stable genes in the desired tissue is required to allow correct result interpretations. The present work aimed at identifying a set of suitable reference genes for expression normalization in blood of HD patients and R6/2 mice. By crossing literature investigation and analysis of microarrays performed on blood of HD patients and healthy subjects, a set of genes was selected and tested by RT-qPCR. Employment of statistical algorithms allowed the identification of the most stable genes in human samples that were than confirmed in R6/2. PPIB, PGK1, ACTB and YWHAZ represent the best possible genes combination, useful to normalize blood transcriptional analysis. To link clinical and preclinical studies, the identified genes were investigated also in blood of R6/2 and wild type mice, confirming that Ppib, Actb and Ywhaz were appropriate for expression normalization. Selected references were subsequently applied to evaluate expression of genes known to be involved in Huntington's pathological progression. This work highlights the importance for correct data normalization to avoid misinterpretation of results, while providing a suitable method to support quantitative gene expression analysis in preclinical and clinical investigations.

Evaluating a Set of Reference Genes for Expression Normalization in Multiple Tissues and Skeletal Muscle at Different Development Stages in Pigs Using Quantitative Real-Time Polymerase Chain Reaction

Gene expression analysis requires the use of reference genes consistently expressed under various conditions. In many cases, however, the commonly used reference genes are not uniformly expressed independently of tissues or environmental conditions. To provide a set of reliable reference genes in pigs, we used quantitative polymerase chain reaction to examine expression of six common reference genes (GAPDH, ACTB, H3F3A, HPRT1, RPL32, and RPS18) in adult tissues and prenatal skeletal muscles at 33, 65, and 90 days postcopulation from Tongcheng (obese-type) and Landrace (lean-type) pigs. The expression stability of these reference genes was evaluated by NormFinder, BestKeeper, and geNorm methods. Our data suggest that the reference genes were expressed variably in different tissues, developmental stages and breeds. RPS18, PRL32, and H3F3A could be used as internal controls to normalize gene expression in pig tissues and developmental skeletal muscle. The combination of internal control genes was necessary for accurate expression normalization. During skeletal muscle development, H3F3A and RPS18 would be the most appropriate combination to normalize gene expression in Tongcheng pigs, whereas the combination of PRL32 and RPS18 would be more suitable in Landrace pigs. In different tissues, the expression of PRL32 and RPS18 was the most consistent, and the combination of three genes (RPL32, RPS18, and H3F3A) is the most suitable for accurate normalization.