Expression Stability Of Reference Genes Research Articles

Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. β-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

Expression of housekeeping genes varies depending on mevalonate pathway inhibition in cancer cells

Statins have anticancer effects and may be used as anticancer agents via drug repositioning. In reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays, the internal reference gene must not be affected by any experimental conditions. As statins exert a wide range of effects on cells by inhibiting the mevalonate pathway, it is possible that statin treatment might alter the expression of housekeeping genes used as internal reference genes, thereby misleading the assessment of obtained gene expression data. Here, we evaluated the expression stability of internal reference genes in atorvastatin-treated cancer cell lines. We treated both statin-sensitive and statin-resistant cancer cell lines with atorvastatin at seven different concentrations and performed RT-qPCR on 15 housekeeping genes whose expression stability was then assessed using five different algorithms. In both statin-sensitive and statin-resistant cancer cell lines, atorvastatin affected the expression of certain internal reference genes in a dose-dependent and cancer cell line-dependent manner; therefore, caution should be exercised when comparing target gene expression between cells. Our findings emphasize the importance of the validation of internal reference genes in gene expression analyses in drug treatment-based cancer research.

Identification of optimal reference genes in golden Syrian hamster with ethanol- and palmitoleic acid-induced acute pancreatitis using quantitative real-time polymerase chain reaction.

Acute pancreatitis (AP) is a severe disorder that leads to high morbidity and mortality. Appropriate reference genes are important for gene analysis in AP. This study sought to study the expression stability of several reference genes in the golden Syrian hamster, a model of AP. AP was induced in golden Syrian hamster by intraperitoneal injection of ethanol (1.35 g/kg) and palmitoleic acid (2 mg/kg). The expression of candidate genes, including Actb, Gapdh, Eef2, Ywhaz, Rps18, Hprt1, Tubb, Rpl13a, Nono, and B2m, in hamster pancreas at different time points (1, 3, 6, 9, and 24 h) posttreatment was analyzed using quantitative polymerase chain reaction. The expression stability of these genes was calculated using BestKeeper, Comprehensive Delta CT, NormFinder, and geNorm algorithms and RefFinder software. Our results show that the expression of these reference genes fluctuated during AP, of which Ywhaz and Gapdh were the most stable genes, whereas Tubb, Eef2, and Actb were the least stable genes. Furthermore, these genes were used to normalize the expression of TNF-α messenger ribonucleic acid in inflamed pancreas. In conclusion, Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

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.

Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides.

Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.

Evaluation of reference genes for miRNA and mRNA normalization in tobacco infected with PVYNTN, PVYN-Wi and PVYZ-NTN strains

This is the first report on identification of the most suitable reference genes for RT-qPCR quantification of miRNA and mRNA in tobacco response to the prevalent recombinant potato virus Y (PVY) strains PVYNTN, PVYN-Wi and the newly identified PVYZ-NTN. Of 10 tested genes, the expression levels of neIF5C, nU2af and nPP2A were the most stable in samples taken from non-inoculated, mock-inoculated, and infected plants at three days post-inoculation (dpi) and 14 dpi. While the homologues of eIF5 were most stably expressed in tobacco in this study and in potato in our previous study (Yin et al., 2021) following inoculation with the same three PVY strains, the homologues of other two genes PP2A and U2af were stably expressed only in tobacco but unstable in potato. The tobacco homologue of PP2A, which was the most stably expressed one in tobacco interaction with PVYNTN, PVYN-Wi and PVYZ-NTN strains in this study, was the least stable one in tobacco interaction with the non-recombinant PVYO strain in a previous study (Baek et al., 2017). This study provides evidence on the influence of host species on expression of housekeeping genes and points out virus strain as a new factor influencing expression stability of reference gene. Caution should be taken when choosing reference genes in gene expression study in Solanaceae hosts response to different PVY strains.

Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model

The choice of appropriate reference genes is essential for correctly interpreting qPCR data and results. However, the majority of animal studies use a single reference gene without any prior evaluation. Therefore, many qPCR results from rodent studies can be misleading, affecting not only reproducibility but also translatability. In this study, the expression stability of reference genes for mRNA and miRNA in archived FFPE samples of 117 C57BL/6JOlaHsd mice (males and females) from 9 colitis experiments (dextran sulfate sodium; DSS) were evaluated and their expression analysis was performed. In addition, we investigated whether normalization reduced/neutralized the influence of inter/intra-experimental factors which we systematically included in the study. Two statistical algorithms (NormFinder and Bestkeeper) were used to determine the stability of reference genes. Multivariate analysis was made to evaluate the influence of normalization with different reference genes on target gene expression in regard to inter/intra-experimental factors. Results show that archived FFPE samples are a reliable source of RNA and imply that the FFPE procedure does not change the ranking of stability of reference genes obtained in fresh tissues. Multivariate analysis showed that the histological picture is an important factor affecting the expression levels of target genes.

Identification and validation of reference genes for qRT-PCR analyses under different experimental conditions in Allium wallichii

Himalayan onion (Allium wallichii) is a perennial bulbous herb with high ornamental value and has long been used as traditional medicines in Nepal and China because of the anti-cancer and anti-microbial activities. Wild Allium wallichii features different flower colors, including purple, pink, deep purple and white. However, little is known about the molecular mechanisms of color formation during A. wallichii flower development stages due to the lack of optimal reference genes. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool for quantifying expression levels of target genes. The accuracy of qRT-PCR analyses is largely dependent on the identification of stable reference genes for data normalization. The stability of reference gene expression may vary with plant species and environmental conditions. The aim of this study was to select stable reference genes for qRT-PCR analyses of target genes at flower development stages, in different flower colors and organs for Allium wallichii. The CDSs of eight potential reference genes (TUB2, ACT1, GAPC, EF1α, UBQ, UBC, SAND and CYP1) were cloned and their stability was evaluated by four programs (Delta Ct, geNorm, NormFinder and BestKeeper), and the results were further integrated into a comprehensive rank by RefFinder. The results showed that TUB2 and GAPC were the most stable two reference genes at different developmental stages of purple- and white-flower genotypes and across all samples. UBC and TUB2 expression was stable at different developmental stages of purple flowers. CYP1 and TUB2 were stably expressed at different developmental stages of white flowers. GAPC and SAND showed the highest rankings in different flower colors. TUB2 and EF1α performed the best in different tissues. ACT1 was the least stable gene in all tested samples. Moreover, DIHYDROFLAVONOL-4-REDUCTASE (DFR) gene that involved in anthocyanin synthesis was used to evaluate the effectiveness of the selected candidates. This study identified the first set of suitable reference genes for qRT-PCR analyses, which will lay the foundation for gene function study in A. wallichii.

Selection and validation of appropriate reference genes for RT–qPCR analysis of Nitraria sibirica under various abiotic stresses

BackgroundNitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT–qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT–qPCR analysis.ResultsIn this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions.ConclusionThis study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis.

Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), β-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), β-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.

Validation of suitable reference genes by various algorithms for gene expression analysis in Isodon rubescens under different abiotic stresses

Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases. Oridonin is one of the main active ingredients, and the route of its molecular biosynthesis remains to be determined. The study of gene expression patterns can provide clues toward the understanding of its biological functions. The selection of suitable reference genes for normalizing target gene expression is the first steps in any quantitative real-time PCR (RT-qPCR) gene expression study. Therefore, validation of suitable reference genes is necessary for obtaining reliable results in RT-qPCR analyses of I. rubescens. Here, 12 candidate reference genes were chosen, and their expression stability in different tissues of I. rubescens and in leaves under different abiotic stresses (NaCl, dehydration, SA, MeJA, and ABA) was evaluated using the ∆Ct, NormFinder, GeNorm, BestKeeper, and RankAggreg statistical tools. Analysis using the comprehensive tools of RankAggreg algorithm showed that GADPH, 18S and eIF were stably expressed in different tissues; UBQ, Apt, and HIS; Cycl, UBQ, and PP2A; GADPH, 18S, and eIF; eIF, UBQ, and PP2A; TUB, Cycl, and UBQ; were the best three candidate reference genes for the samples of Dehydration, NaCl, SA, MeJA, and ABA treatment, respectively. While for the concatenated sets of ND (NaCl and dehydration) and SMA (SA, MeJA, and ABA), UBQ, HIS, and TUA; UBQ, eIF and Apt were the three appropriate candidate reference genes, respectively. In addition, the expression patterns of HMGR in different tissues and under different treatments were used to confirm the reliability of the selected reference genes, indicating that the use of an inappropriate reference gene as the internal control will cause results with a large deviation. This work is the first study on the expression stability of reference genes in I. rubescens and will be particularly useful for gene functional research in this species.

Validation of quantitative real-time PCR reference genes and spatial expression profiles of detoxication-related genes under pesticide induction in honey bee, Apis mellifera.

Recently, pesticides have been suggested to be one of the factors responsible for the large-scale decline in honey bee populations, including colony collapse disorder. The identification of the genes that respond to pesticide exposure based on their expression is essential for understanding the xenobiotic detoxification metabolism in honey bees. For the accurate determination of target gene expression by quantitative real-time PCR, the expression stability of reference genes should be validated in honey bees exposed to various pesticides. Therefore, in this study, to select the optimal reference genes, we analyzed the amplification efficiencies of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAD1a) and their expression stability values using four programs (geNorm, NormFinder, BestKeeper, and RefFinder) across samples of five body parts (head, thorax, gut, fat body, and carcass) from honey bees exposed to seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin). Among these five candidate genes, a combination of RAD1a and RPS18 was suggested for target gene normalization. Subsequently, expression levels of six genes (AChE1, CYP9Q1, CYP9Q2, CYP9Q3, CAT, and SOD1) were normalized with a combination of RAD1a and RPS18 in the different body parts from honey bees exposed to pesticides. Among the six genes in the five body parts, the expression of SOD1 in the head, fat body, and carcass was significantly induced by six pesticides. In addition, among seven pesticides, flupyradifurone statistically induced expression levels of five genes in the fat body.

Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms.

Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determine the expression stability of commonly used RGs in mouse skeletal muscle tissue. The expression pattern of eight RGs (ACTB, GAPDH, HPRT, YWHAZ, B2M, PPIA, TUBA and 18S) were evaluated by RT-qPCR in different sample groups classified based on genetic background, muscle tissue type, and growth stage, as well as in a C2C12 myoblast cell line model. Five computational programs were included in the study (comparative ΔCq value, NormFinder, BestKeeper, geNorm, RefFinder) to evaluate the expression stability of RGs. Furthermore, the normalization effects of RGs in soleus (SOL) and gastrocnemius (GAS) muscle tissue were evaluated. Collectively, ACTB, HPRT and YWHAZ were shown to be the most stable RGs, while GADPH and 18S were the least stable. Therefore, the combined use of ACTB, HPRT and YWHAZ is recommended for the normalization of gene expression results in experiments with murine skeletal muscle. The results discussed herein provide a foundation for gene expression analysis by RT-qPCR in mammalian skeletal muscle.

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.

Reference Genes Selection of Gymnosporangium yamadae during the Interaction with Apple Leaves.

Apple rust disease caused by Gymnosporangium yamadae is the one of the major threats to the development of the apple industry in China, but the pathogenic molecular mechanism of the disease remains unclear. It is imperative to screen out appropriate reference genes during the interaction between G. yamadae and apple leaves to analyze the gene expression patterns during the pathogenesis of G. yamadae. ACT, EF1, EF2, GAPDH, 40S, 60S, α-TUB, β-TUB and UBCE3 were selected as candidate reference genes based on the transcriptomic dataset of G. yamadae. The expression levels were tested by real-time quantitative PCR during time-course infection of apple leaves and the expression stabilities were evaluated by △Ct method as well as by three software (NormFinder, geNorm and BestKeeper) and one web-based analysis software (RefFinder). The expression stability of the candidate reference genes was further validated by using the effector candidate gene Cluster-3395.48660 as the target gene in RT-qPCR. According to the results by △Ct and BestKeeper, 40S, EF2 and EF1 were the most stable reference genes, while EF1, EF2 and GAPDH were the most stable reference genes based on the NormFinder analysis result. The geNorm recommended the most stable genes EF1, EF2 and α-TUB as reference genes. Comprehensive analysis results of the RefFinder indicated EF1, EF2 and α-TUB were the most suitable genes. Based on these results, EF1, EF2 and α-TUB were considered as reference genes for analyzing the gene expression profiles of Cluster-3395.48660 in different infection stages, and the results were consistent with the transcriptome data. All the results suggest that the combination of EF1, EF2 and α-TUB proved to be acceptable reference genes during the interaction between G. yamadae and apple leaves.

P-517 Validating reference gene expression stability in human ovarian follicles, oocytes, cumulus cells, ovarian medulla, and ovarian cortex tissue

Abstract Study question How does the expression stability of commonly used reference genes (RG) vary in different human ovarian cells and tissues? Summary answer The RG stability differed among human ovarian cell types and tissues, but an RG with high stability was identified for each cell and tissue type. What is known already The expression of RGs used to normalize RT-qPCR may vary between different tissues, cell types, and experimental conditions. Hence, selecting the most appropriate RGs is critical in any experimental design to interpret data generated by RT-qPCR with the best accuracy. Human ovarian cells are phenotypically very different and often only available in limited amounts. In several animal species RG expression stability has been validated in oocytes and other ovarian cells, however, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. Study design, size, duration This is an experimental study performed at a university hospital from January 2021 to September 2021. Participants/materials, setting, methods The freely available NormFinder software was used to analyze the expression stability of five commonly used RGs (GAPDH, B2M, RPLP0, ACTB, and PPIA) in human oocytes (n = 160), preantral follicles (n = 160), cumulus cells (n = 13), ovarian medulla (n = 8), and ovarian cortex tissue (n = 60). Samples were collected from 29 patients (aged 28 years on average; range 14–36) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Main results and the role of chance The Normfinder software identified ACTB as the best RG for oocytes and cumulus cells; and B2M for medulla tissue and isolated follicles. Overall, comparisons of the cycle threshold (Ct) values demonstrated a wide variation among the RGs within the same group of samples (P < 0.05), especially for oocytes and preantral follicles with normalized RNA. The genes ACTB and RPLP0 showed the highest levels of expression and PPIA the lowest levels of expression in all types of samples, except for cortex tissue, where PPIA had the highest level of expression and B2M the lowest. These results infer that different results could be obtained when using different RGs for data normalization. The combination of two RGs only marginally increased stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For cultured ovarian cortex culture, GAPDH or ACTB were found to be the most stable genes depending on culture conditions. Limitations, reasons for caution Since human oocytes are scarce and contain a small amount of RNA, only five RGs were evaluated. Moreover, only germinal vesicle (GV) oocytes and preantral follicles (60-80 µm) were tested. Future studies may validate other genes and investigate the effect of oocyte maturation and follicle growth on RG stability. Wider implications of the findings Our findings highlight the importance of validating RGs for each cell type or tissue and culture condition. Hence, our results can be of use as guidance for future studies involving gene expression analyses in human ovarian cells and tissues, including oocytes and preantral follicles. Trial registration number Not applicable

Selection and Validation of Reference Genes for RT-qPCR Analysis in Tibetan Medicinal Plant Saussurea Laniceps Callus under Abiotic Stresses and Hormone Treatments.

Real-time quantitative PCR (RT-qPCR) is an important technique for studying gene expression analysis, but accurate and reliable results depend on the use of a stable reference gene. This study proposes to test the expression stability of candidate reference genes in the callus of Saussurea laniceps, a unique Tibetan medicinal plant. Based on the S. laniceps callus transcriptome, eleven candidate reference genes, including TUA2, TUB3, TUB8, TIF3B1, TIF3H1, ELF5A, PP2AA2, UEV1D, UBL5, UBC36, and SKIP1), were validated for RT-qPCR normalization in the callus under abiotic stress (salt, cold, and UV) and hormone treatments (abscisic acid, MeJA, and salicylic acid). The stability of the candidate genes was evaluated in all the samples of S. laniceps. Comprehensive analysis of all samples showed that the best reference genes were UBC36 and UBL5. ELF5A and TIF3B1 were ranked as the most stable genes in the sample sets under abiotic stress. For hormone stimulation, UBC36 and TIF3H1 genes had the best stability. This study provides useful guidelines and a starting point for reference gene selection for expression analysis using RT-qPCR techniques in S. laniceps.

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue.

Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress.

Bradysia odoriphaga (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling B. odoriphaga. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in B. odoriphaga, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

Selection of reference genes suitable for normalization of qRT‐PCR data in spotted sea bass ( Lateolabrax maculatus ) under conditions of pathogen infections

The aim of this study was to select the most suitable reference genes for qRT-PCR of spotted sea bass (Lateolabrax maculatus) under different pathogen infection conditions. A total of 10 candidate reference genes were selected; they included RPL19, RPL7, RPL13, TUGB, 18S rRNA, CYTB5, eIF5A, ACTB, GAB2 and GAPDH. Transcript levels of these candidate reference genes were examined using qRT-PCR analysis in 11 tissues (heart, liver, stomach, pectoral fin, intestine, muscle, brain, skin, gill, head kidney and spleen). Four algorithms, geNorm, NormFinder, BestKeeper and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. RPL19 was the most stable gene in different tissues in L. maculatus under normal conditions according to the overall ranking of the four algorithms. GAPDH was the most unstable gene in different tissues. GAB2 was the most stable gene in both head kidney and liver in L. maculatus under V. harveyi infection; RPL19 and RPL13 were the most stable genes in head kidney and liver in L. maculatus under S. iniae infection respectively; RPL19 and GAB2 were the most stable genes in head kidney and liver in L. maculatus under RGV infection respectively; RPL7 and RPL19 were the most stable genes in head kidney and spleen in L. maculatus under ISKNV infection respectively. GAPDH was the most unstable gene in immune tissues in L. maculatus under pathogen infections. The present study provides the first validated reference genes for accurate data normalization in transcript profiling in L. maculatus infected by bacteria (V. harveyi and S. iniae) and virus (ISKNV and RGV), which will be indispensable for further gene expression in this economically valuable marine teleost.