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The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus. [ABSTRACT FROM AUTHOR]

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-Nacetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance. [ABSTRACT FROM AUTHOR]

Bumblebees (B. terrestris) play a crucial role as highly efficient biological agents in commercial pollination. Understanding the molecular mechanisms governing their adaptation to diverse seasonal environments may pave the way for effective management strategies in the future. With the burgeoning advancement in post-genetic studies focusing on B. terrestris, there is a critical need to normalize quantitative real-time PCR (qRT-PCR) data using suitable reference genes. To address this necessity, we employed RefFinder, a software-based tool, to assess the suitability of several candidate endogenous control genes, including actin (ACT), arginine kinase (AK), elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate (GAPDH), phospholipase (PLA2), and ribosomal proteins (S18, S28). These genes were evaluated for their efficacy as biological endogenous controls by examining their expression patterns across various environmental conditions corresponding to different seasons (Spring, Summer, Autumn, Winter) and tissues (ovary, fat body, thorax, head) in bumblebees. Moreover, the study investigated the significance of selecting appropriate reference genes for three key genes involved in the juvenile hormone (JH) signaling pathways: Krüppel homolog 1 (Kr-h1), methyl farnesoate epoxidase (MFE), and Vitellogenin (Vg). Our research identifies specific genes suitable for normalization in B. terrestris, thereby offering valuable insights into gene expression and functional metabolic genetics under varying seasonal conditions. This catalog of reference genes will serve as a valuable resource for future research endeavors.

Background: Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. Methods: This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. Results: After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. Conclusion: The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues. [ABSTRACT FROM AUTHOR]

The Gill's mealybug, Ferrisia gilli Gullan, (Hemiptera: Pseudococcidae) has emerged as a major pest of pistachio in California. Because F. gilli is only relatively recently described, there are no validated reference genes to normalize the expression data from real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) in this species. We selected and validated 8 commonly used reference genes (RPS8 , TBP , UBQE2 , RPL7 , RPL5 , RPL40 , RPLP1 , and HEL) for expression stability in F. gilli. These genes were evaluated in 5 different geographical populations of F. gilli collected from organic and conventionally grown pistachio orchards. Candidate reference genes were also evaluated in F. gilli fed with 4 plant hosts: pistachio, almond, grapes, and lima beans. The stability of candidate genes was analyzed using 4 software algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Three genes RPS8 , RPL40 , and RPL7 encoding for ribosomal proteins were identified as the most stable across the treatments and thus were recommended for normalizing the qRT-PCR data. These findings will support resistance monitoring, molecular toxicology, and functional genomics research in F. gilli. [ABSTRACT FROM AUTHOR]

Abstract Background Nitraria 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. Results In 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. Conclusion This 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.

Pseudomonas aeruginosa is an opportunistic human pathogen notorious for its remarkable capacity of multi-drug resistance, and has become one of the most important model bacteria in clinical bacteriology research. Quantitative real-time PCR is a reliable method widely used in gene expression analysis, for which the selection of a set of appropriate housekeeping genes is a key prerequisite for the accuracy of the results. However, it is easy to overlook that the expression level of housekeeping gene may vary in different conditions, especially in the condition of molecular microbiology assays, where tested strains are generally cultured under the pre-set antibiotic selection pressures, and how this affects the stability of commonly used housekeeping genes remains unclear. In this study, the expression stability of ten classic housekeeping genes (algD, gyrA, anr, nadB, recA, fabD, proC, ampC, rpoS, and rpsL) under the pressure of eight laboratory commonly used antibiotics (kanamycin, gentamycin, tetracycline, chloramphenicol, hygromycin B, apramycin, tellurite, and zeocin) were tested. Results showed that the stability of housekeeping gene expression was indeed affected by the types of antibiotics added, and of course the best reference gene set varied for different antibiotics. This study provides a comprehensive summary of the effects of laboratory antibiotics on the stability of housekeeping genes in P. aeruginosa, highlighting the necessity to select housekeeping genes according to the type of antibiotics used in the initial stage of experiment. [ABSTRACT FROM AUTHOR]

Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai–Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats. [ABSTRACT FROM AUTHOR]

Abstract Background Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. Results As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. Conclusions We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.

Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (ADF2, CYB5, iPGAM, SCL13, TRXL3-3, and VHA-H) with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend CYB5 to be used as RG for the leaf colour experiment and TRXL3-3 for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in Lactuca spp. under the described conditions. [ABSTRACT FROM AUTHOR]

Background: Nitraria 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. Results: In 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. Conclusion: This 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. [ABSTRACT FROM AUTHOR]

Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species. [ABSTRACT FROM AUTHOR]

Eichhornia crassipes is a notorious invasive aquatic weed, causing enormous ecological and economic losses worldwide. However, it has great potential in agriculture, industry, medical care, and other areas. While being such an important plant, it is poorly understood from the molecular perspective. Aiming to select suitable reference genes for gene expression quantification in E. crassipes, this study favors future research at the molecular level. In this work, 12 candidate reference genes were selected. Their expression stability in samples of different tissues, samples treated with various hormones, samples supplied with different levels of phosphorus (P), and pooled samples, were analyzed using GeNorm, NormFinder, BestKeeper, and RefFinder. Meanwhile, the optimal number of reference genes was calculated by GeNorm. The results showed that eIF and ElF1a were the two most stable reference genes in all samples and in tissue samples. In response to hormone treatments, Actin and eIF are the best choices of internal controls. In the case of P treatments, TUA and H2A are recommended to be used as reference genes. Overall, results from this work suggest different reference genes should be applied in qRT-PCR on E. crassipes, according to the specific experimental setup. [ABSTRACT FROM AUTHOR]

Background: Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. Results: As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. Conclusions: We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions. [ABSTRACT FROM AUTHOR]

Gene expression analysis largely improves our understanding of the molecular basis underpinning various plant biological processes. Stable reference genes play a foundational role during the normalization of gene expression levels. However, until now, there have been few reference genes suitable for ginger reverse transcription-quantitative PCR (RT-qPCR) research. In this study, 29 candidate reference genes with stable expression patterns across multiple ginger tissues and 13 commonly used reference genes were selected to design RT-qPCR primers. After amplification specificity validation, 32 candidates were selected and further evaluated by RT-qPCR using samples from various organs subjected to NaCl, drought, heat, waterlogging, and chilling stress. Four strategies, including delta-CT, BestKeeper, geNorm, and NormFinder, were used to rank the stability of reference genes, and the ranks produced by these four strategies were comprehensively evaluated by RefFinder to determine the final rank. Overall, the top three stability reference genes indicated by RefFinder were RBP > ATPase > 40S_S3. Their expression pattern correlation analysis showed that the coefficients among each pair of RBP , ATPase , and 40S_S3 were larger than 0.96, revealing consistent and stable expression patterns under various treatments. Then, the expression of three pathogenesis-related (PR) genes and seven MYB genes in rhizomes during postharvest storage and subjected to pathogen infection was normalized by RBP , ATPase , 40S_S3 , RBP and ATPase , ATPase and 40S-S3 , and RBP and 40S-S3. The results showed that PR and MYB genes were induced by postharvest deterioration and pathogen infection. The correlation coefficients of RBP / ATPase , RBP / 40S_S3 , ATPase / 40S_S3 , RBP and ATPase/ATPase and 40S-S3 , RBP and ATPase/RBP and 40S-S3 , and ATPase and 40S-S3/RBP and 40S-S3 were 0.99, 0.96, 0.99, 0.99, 1.00, and 1.00, respectively, which confirmed the stability of these three reference genes in postharvest biology studies of ginger. In summary, this study identified appropriate reference genes for RT-qPCR in ginger and facilitated gene expression studies under biotic and abiotic stress conditions. [ABSTRACT FROM AUTHOR]

Gene expression analysis largely improves our understanding of the molecular basis underpinning various plant biological processes. Stable reference genes play a foundational role during the normalization of gene expression levels. However, until now, there have been few reference genes suitable for ginger reverse transcription-quantitative PCR (RT-qPCR) research. In this study, 29 candidate reference genes with stable expression patterns across multiple ginger tissues and 13 commonly used reference genes were selected to design RT-qPCR primers. After amplification specificity validation, 32 candidates were selected and further evaluated by RT-qPCR using samples from various organs subjected to NaCl, drought, heat, waterlogging, and chilling stress. Four strategies, including delta-CT, BestKeeper, geNorm, and NormFinder, were used to rank the stability of reference genes, and the ranks produced by these four strategies were comprehensively evaluated by RefFinder to determine the final rank. Overall, the top three stability reference genes indicated by RefFinder were RBP > ATPase > 40S_S3. Their expression pattern correlation analysis showed that the coefficients among each pair of RBP, ATPase, and 40S_S3 were larger than 0.96, revealing consistent and stable expression patterns under various treatments. Then, the expression of three pathogenesis-related (PR) genes and seven MYB genes in rhizomes during postharvest storage and subjected to pathogen infection was normalized by RBP, ATPase, 40S_S3, RBP and ATPase, ATPase and 40S-S3, and RBP and 40S-S3. The results showed that PR and MYB genes were induced by postharvest deterioration and pathogen infection. The correlation coefficients of RBP/ATPase, RBP/40S_S3, ATPase/40S_S3, RBP and ATPase/ATPase and 40S-S3, RBP and ATPase/RBP and 40S-S3, and ATPase and 40S-S3/RBP and 40S-S3 were 0.99, 0.96, 0.99, 0.99, 1.00, and 1.00, respectively, which confirmed the stability of these three reference genes in postharvest biology studies of ginger. In summary, this study identified appropriate reference genes for RT-qPCR in ginger and facilitated gene expression studies under biotic and abiotic stress conditions.

The reverse transcription quantitative real‐time PCR (RT‐qPCR) is widely preferred tool for expression analysis of target gene at mRNA level. However, in order to obtain significant biological comparison, data normalization to a stable endogenous internal control gene also called reference gene (RG) is of utmost importance. In the present study, we evaluated the expression stability of four commonly used housekeeping genes, that is beta actin, β‐actin; glyceraldehyde‐3‐phosphate dehydrogenase, GAPDH; elongation factor‐1 alpha, EF‐1α and 18S ribosomal RNA, 18S for qPCR data normalization. This study was conducted for two varying physiological (Group I: fed vs. non‐fed and Group II: earthen pond vs. fibre reinforced plastic tank reared) and one pathological condition (Group III: Aeromonas hydrophila injected vs. PBS injected) in seven different tissues of Indian catfish, Clarias magur, an economically valuable food fish of South‐East Asia. Tissue wise expression stability analysis using web‐based comprehensive tool Reffinder revealed that EF‐1α was the most stable RG in kidney, liver, spleen and gills of group I; spleen, intestine, muscle and brain in group II; and kidney, liver, intestine, muscle and brain in group III. β‐actin was the most stable RG in intestine of group I; liver and brain in group II; spleen and gill in group III. 18S was the most stable RG in muscle and brain of group I; kidney and gill in group II. GAPDH was the least stable RG among four selected genes under all the experimental conditions. These findings on selection of RGs will lead to precise interpretation of relative gene expression in different tissues by RT‐qPCR in C. magur in the context of physiological studies and bacterial infection. [ABSTRACT FROM AUTHOR]

The quantitative real-time polymerase chain reaction (qRT-PCR) is the most sensitive and commonly used technique for gene expression studies in biological systems. However, the reliability of qRT-PCR results depends on the selection of reference gene(s) for data normalization. Horse gram (Macrotyloma uniflorum) is an important legume crop on which several molecular studies have been reported. However, the stability of reference genes has not been evaluated. In the present study, nine candidate reference genes were identified from horse gram RNA-seq data and evaluated in two horse gram genotypes, HPK4 and HPKM317 under six abiotic stresses viz. cold, drought, salinity, heat, abscisic acid and methyl viologen-induced oxidative stress. The results were evaluated using geNorm, Bestkeeper, Normfinder and delta-delta Ct methods and comprehensive ranking was assigned using RefFinder and RankAggreg software. The overall result showed that TCTP was one of the most stable genes in all samples and in genotype HPK4, while in HPKM317 profilin was most stably expressed. However, PSMA5 was identified as least stable in all the experimental conditions. Expression of target genes dehydrin and early response to dehydration 6 under drought stress was also validated using TCTP and profilin for data normalization, either alone or in combination, which confirmed their suitability for qRT-PCR data normalization. Thus, TCTP and profilin genes may be used for qRT-PCR data normalization for molecular and genomic studies in horse gram. [ABSTRACT FROM AUTHOR]

Paederus fuscipes is a medically and agriculturally important insect all over the world. P. fuscipes cause not only a skin condition but is also an aggressive predator in different agro-ecosystems. Prior to performing RT-qPCR under a variety of conditions to investigate the function of target genes in P. fuscipes, it is essential to screen reference genes. However, no P. fuscipes reference gene(s) has yet been discovered. Using the RefFinder software package, which includes ΔCt, geNorm, NormFinder, and BestKeeper, we evaluated the stability of seven housekeeping genes of P. fuscipes under five conditions (developmental stage, diet, temperature, tissue and sex). During the RT-qPCR analysis, geNorm software was used to evaluate pairwise variation in order to identify the most appropriate number of reference genes. The results revealed that for developmental stages RPL-13, EF1A and β-tubulin; for sex-related experiments β-tubulin and Actin, for tissue-related experiments PRS-18, 18S and RPL-13; for temperature-related experiments β-tubulin and PRL-13 and diet-related experiments, β-tubulin and PRS-18 are suitable reference genes. Furthermore, the associated HSP-70 (as the reporter gene) expression patterns differed significantly when the three most stable and three least stable reference genes were selected in different temperature treatments. This is the first time that a complete list of standardized reference genes has been given for P. fuscipes to use in an RT-qPCR study, which could be helpful for potential functional studies of target genes in P. fuscipes.

Background: Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches., Aim: To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR., Methods: We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 ( RPLP0 ), TATA-binding protein ( TBP ), GAPDH , actin beta ( ACTB ), tubulin ( TUB ), aminolevulinic acid synthase 1 ( ALAS1 ), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta ( YWHAZ ), eukaryotic translational elongation factor 1 alpha ( EF1a ), succinate dehydrogenase complex, subunit A, flavoprotein ( SDHA ), and beta-2-microglobulin ( B2M )] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups., Results: All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking., Conclusion: For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers., Competing Interests: Conflict-of-interest statement: All authors have no conflicts of interest to declare., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species.

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI, dnaG, and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytvI, dnaG, and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI , dnaG , and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytv I, dnaG , and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium. [ABSTRACT FROM AUTHOR]

Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (ACTB , BACH1 , B2M , GAPDH , HMBS , PGK1 , PPIA , PPIB , RPLP0 , RPL19 , RPS9 , RPS15 , RPS28 , and UXT) in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that PPIB, BACH1, ACTB , and PPIA are the most suitable RGs, while RPLP0, GAPDH and RPS15 are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations. • Systematically assessed 14 potential reference genes in sheep/goat PBMCs to find stable qPCR normalizers. • Four algorithms, namely, geNorm, NormFinder, BestKeeper, and Delta Ct were used to rank the genes. • PPIB , BACH1 , ACTB , and PPIA were identified as the most stable reference genes. • RPLP0 , GAPDH and RPS15 were the least stable genes in the PBMCs of sheep and goat. [ABSTRACT FROM AUTHOR]

• A systematic assessment of 18 potential reference genes (RGs) in 10 different ovine tissues was carried out to identify internal control genes for qPCR analysis. • Four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), were employed to rank the stability of these RGs. • RefFinder was used for a comprehensive ranking, identifying ACTB, PPIB, BACH1 , and B2M as the most stable RGs. • RPS9, RPS15 , and PGK1 showed variable expression across different tissues. • Validation was performed using qPCR analysis of four target genes in the skin from two sheep breeds. Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB , BACH1 , B2M , GAPDH , HMBS , HPRT1 , PGK1 , PPIA , PPIB , RPLP0 , RPL19 , RPS9 , RPS15 , RPS28 , SDHA , TBP , UXT , and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1 , and B2M emerged as the most stable RGs, while RPS9, RPS15 , and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1 , and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1 , and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues. [ABSTRACT FROM AUTHOR]

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH, TATA, RPS18 (for starvation-induced stress), TATA, RPS6, and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions.

Rhopalosiphum padi (L.) (Hemiptera: Aphididae) is an important cosmopolitan pest in cereal crops. Reference genes can significantly affect qRT-PCR results. Therefore, selecting appropriate reference genes is a key prerequisite for qRT-PCR analyses. This study was conducted to identify suitable qRT-PCR reference genes in R. padi. We systematically analyzed the expression profiles of 11 commonly used reference genes. The ΔCt method, the BestKeeper, NormFinder, geNorm algorithms, and the RefFinder online tool were used to evaluate the suitability of these genes under diverse experimental conditions. The data indicated that the most appropriate sets of reference genes were β-actin and GAPDH (for developmental stages), AK and TATA (for populations), RPS18 and RPL13 (for tissues), TATA and GAPDH (for wing dimorphism), EF-1α and RPS6 (for antibiotic treatments), GAPDH and β-actin (for insecticide treatments), GAPDH , TATA , RPS18 (for starvation-induced stress), TATA , RPS6 , and AK (for temperatures), and TATA and GAPDH (for all conditions). Our study findings, which revealed the reference genes suitable for various experimental conditions, will facilitate the standardization of qRT-PCR programs, while also improving the accuracy of qRT-PCR analyses, with implications for future research on R. padi gene functions. [ABSTRACT FROM AUTHOR]

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica , have been tremendously limited. In this study, ten internal reference genes ( ACT , ARF1 , AK , EF1α , GAPDH , α-TUB , RPL6 , RPL13 , RPS3 and RPS18 ) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase ( MsUAP ), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica , such as development, reproduction, sex differentiation, cold and heat resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shen, Tang, Li, Zhu, Li, Deng, Zhai, Wu and Yan.)

Abstract Background Sugarcane (Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. Results In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots. Conclusion Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.

Bumblebees ( B. terrestris ) play a crucial role as highly efficient biological agents in commercial pollination. Understanding the molecular mechanisms governing their adaptation to diverse seasonal environments may pave the way for effective management strategies in the future. With the burgeoning advancement in post-genetic studies focusing on B. terrestris , there is a critical need to normalize quantitative real-time PCR (qRT-PCR) data using suitable reference genes. To address this necessity, we employed RefFinder, a software-based tool, to assess the suitability of several candidate endogenous control genes, including actin ( ACT ), arginine kinase ( AK ), elongation factor 1 alpha ( EF1 ), glyceraldehyde-3-phosphate ( GAPDH ), phospholipase ( PLA2 ), and ribosomal proteins ( S18 , S28 ). These genes were evaluated for their efficacy as biological endogenous controls by examining their expression patterns across various environmental conditions corresponding to different seasons (Spring, Summer, Autumn, Winter) and tissues (ovary, fat body, thorax, head) in bumblebees. Moreover, the study investigated the significance of selecting appropriate reference genes for three key genes involved in the juvenile hormone (JH) signaling pathways: Krüppel homolog 1 ( Kr-h1 ), methyl farnesoate epoxidase ( MFE ), and Vitellogenin ( Vg ). Our research identifies specific genes suitable for normalization in B. terrestris , thereby offering valuable insights into gene expression and functional metabolic genetics under varying seasonal conditions. This catalog of reference genes will serve as a valuable resource for future research endeavors.

Alexandrium catenella is a cosmopolitan harmful algal bloom (HAB) forming dinoflagellate. To comprehend its mechanisms of bloom formation, gene expression analysis is indispensable. Quantitative real-time reverse transcription PCR (qRT-PCR) is an ideal method for swift and precise quantification of gene expression analysis that greatly relies on selection of apposite reference genes for data normalization. To date, limited studies have focused on the screening of reference genes in dinoflagellates. The unavailability of valid reference gene for normalizing poses hindrances in the appliance of qRT-PCR to the HAB forming group. The work presented here evaluated 12 reference genes for their expression stability using qRT-PCR under diverse nutritional conditions together with high light. Statistical algorithm such as RefFinder was used that analyze data using geNorm and NormFinder, comparative delta-CT method along with a combination approach that declares comprehensive ranking contingent upon the geometric mean of the results procured from other methods. Comprehensive analysis across all condition by geNorm showed ACT, IPP, and GAPDH as genes possessing highest stability followed by Tubα and ICDH. Comprehensive analysis by Normfinder declared that IPP is the most stable gene followed by ACT, CYC, GAPDH, and GTEF. Combination approach using comparative delta CT, geNorm and NormFinder analysis programs through RefFinder as well as ranking by standard deviation of delta CT declared IPP as the most stable gene followed by ACT and GAPDH. IPP, ACT, and GAPDH represented the top 4 listed stable reference genes among all the analysis methods used. The results of pairwise variation by geNorm showed that V2/V3 value under all the tested conditions was below the cut-off value of 0.15 which shows that two genes are sufficient for normalization. Our results in accord with other widely conducted studies emphasize the significance of reference gene validation in precise experimental setup before appliance to avoid serious misinterpretation of the results. It is highly expected that the procedures and outcome of the report may be helpful for future studies in the gene expression analysis of A. catenella and to screen out reference genes for other algae. [ABSTRACT FROM AUTHOR]

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin, GAPDH, RPL13, RPL6, RPL32, RPS18, and ATPB, were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder, which integrates four different analytical tools (Normfinder, geNorm, the ΔCt method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1, varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata.

When performing expression analysis either for coding RNA (e.g., mRNA) or non-coding RNA (e.g., miRNA), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used method. To normalize these data, one or more stable endogenous references must be identified. RefFinder is an online web-based tool using four almost universally used algorithms for assessing candidate endogenous references-delta-Ct, BestKeeper, geNorm, and Normfinder. However, the online interface is presently cumbersome and time consuming. We developed an R package, RefSeeker, which performs easy and straightforward RefFinder analysis by enabling raw data import and calculation of stability from each of the algorithms and provides data output tools to create graphs and tables. This protocol uses RefSeeker R package for fast and simple RefFinder stability analysis. Key features Perform stability analysis using five algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder. Identification of endogenous references for normalization of RT-qPCR data. Create publication-ready graphs and tables output. Step-by-step guide dialog window for novice R users., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a momentous technique for quantifying expression levels of the targeted genes across various biological processes. Selection and validation of appropriate reference genes for RT-qPCR analysis are a pivotal precondition for reliable expression measurement. Henosepilachna vigintioctopunctata is one of the most serious insect pests that attack Solanaceae plants in Asian countries. Recently, the transcriptomes of H. vigintioctopunctata were sequenced, promoting gene functional studies of this insect pest. Unfortunately, the reference genes for H. vigintioctopunctata have not been selected and validated. Here, a total of 7 commonly used reference genes, namely, Actin , GAPDH , RPL13 , RPL6 , RPL32 , RPS18 , and ATPB , were selected and assessed for suitability under four experimental conditions, namely, developmental stage, tissue, temperature, and host plant, using RefFinder , which integrates four different analytical tools (Normfinder , geNorm , the Δ Ct method, and BestKeeper). The results displayed that RPL13 and RPS18 were the best suitable reference genes for each experimental condition. The relative transcript levels of 2 target genes, lov and TBX1 , varied greatly according to normalization with the two most- and least-suited reference genes. Our results will be helpful for improving the accuracy of the RT-qPCR analysis for future functional investigations of target gene expression in H. vigintioctopunctata. [ABSTRACT FROM AUTHOR]

Study on expression of genes for the traits associated with hypoxia tolerance during the germination demands robust choice of reference genes for transcript data normalization and gene validation through real-time quantitative polymerase chain reaction (RT-qPCR). However, reliability and stability of reference genes across different rice germplasms under hypoxic condition have not been accessed yet. Stability performance of reference genes such as eukaryotic elongation factor 1 α (eEF1α), ubiquitin 10 (UBQ10), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18SrRNA), 25S ribosomal RNA (25SrRNA), β-tublin (β-TUB), actin11 (ACT11), ubiquitin C (UBC), eukaryotic elongation factor 4 α (eIF4α), and ubiquitin5 (UBQ5) was accessed through statistical algorithms like geNorm, NormFinder, Comparative ΔCt method, BestKeeper, and RefFinder in three rice germplasms (KHO, RKB, and IR-64) with varied level of tolerance to hypoxic condition during germination. Among all genes used, OsGAPDH was found to be the most suitable reference gene under hypoxic condition. The performance of the highest-ranking reference gene (OsGAPDH) in terms of stability based on statistical algorithms was further validated for its reliability and stability through RT-qPCR with hypoxia-induced target gene OsTTP7. The identified stable housekeeping gene could be used as internal control for gene expression analysis in rice under hypoxia. [ABSTRACT FROM AUTHOR]

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. [ABSTRACT FROM AUTHOR]

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L . japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L . japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L . japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT , 18S rRNA , and RPL13 during different stages; AK , RPL13 , and TBP among sexes; EF1A , PPI , and RPL27 in different tissues, and EF1A , RPL13 , and PPI in adults fed on different diets. Moreover, the expression profile of a target gene ( odorant receptor 1 , OR1 ) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L . japonica and will benefit subsequent genomics and functional genomics research on this natural enemy. [ABSTRACT FROM AUTHOR]

Many studies use strategies that allow for the identification of a large number of genes expressed in response to different stress conditions to which the plant is subjected throughout its cycle. In order to obtain accurate and reliable results in gene expression studies, it is necessary to use reference genes, which must have uniform expression in the majority of cells in the organism studied. RNA isolation of leaves and expression analysis in real-time quantitative polymerase chain reaction (RT-qPCR) were carried out. In this study, nine candidate reference genes were tested, actin 11 ( ACT11), ubiquitin conjugated to E2 enzyme ( UBC-E2), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), beta tubulin ( β-tubulin), eukaryotic initiation factor 4α ( eIF-4α), ubiquitin 10 ( UBQ10), ubiquitin 5 ( UBQ5), aquaporin TIP41 ( TIP41-Like) and cyclophilin, in two genotypes of rice, AN Cambará and BRS Querência, with different levels of soil moisture (20%, 10% and recovery) in the vegetative (V5) and reproductive stages (period preceding flowering). Currently, there are different softwares that perform stability analyses and define the most suitable reference genes for a particular study. In this study, we used five different methods: geNorm, BestKeeper, ΔCt method, NormFinder and RefFinder. The results indicate that UBC-E2 and UBQ5 can be used as reference genes in all samples and softwares evaluated. The genes β-tubulin and eIF-4α, traditionally used as reference genes, along with GAPDH, presented lower stability values. The gene expression of basic leucine zipper ( bZIP23 and bZIP72) was used to validate the selected reference genes, demonstrating that the use of an inappropriate reference can induce erroneous results. [ABSTRACT FROM AUTHOR]

Background: Sugarcane (Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. Results: In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and Ref-Finder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots. Conclusion: Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions. [ABSTRACT FROM AUTHOR]

Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat and abscisic acid) and tissues (leaves, roots, seedlings, panicle and mature green seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A;PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes.

Pseudomonas aeruginosa is an opportunistic human pathogen notorious for its remarkable capacity of multi-drug resistance, and has become one of the most important model bacteria in clinical bacteriology research. Quantitative real-time PCR is a reliable method widely used in gene expression analysis, for which the selection of a set of appropriate housekeeping genes is a key prerequisite for the accuracy of the results. However, it is easy to overlook that the expression level of housekeeping gene may vary in different conditions, especially in the condition of molecular microbiology assays, where tested strains are generally cultured under the pre-set antibiotic selection pressures, and how this affects the stability of commonly used housekeeping genes remains unclear. In this study, the expression stability of ten classic housekeeping genes ( algD, gyrA, anr, nadB, recA, fabD, proC, ampC, rpoS, and rpsL ) under the pressure of eight laboratory commonly used antibiotics (kanamycin, gentamycin, tetracycline, chloramphenicol, hygromycin B, apramycin, tellurite, and zeocin) were tested. Results showed that the stability of housekeeping gene expression was indeed affected by the types of antibiotics added, and of course the best reference gene set varied for different antibiotics. This study provides a comprehensive summary of the effects of laboratory antibiotics on the stability of housekeeping genes in P. aeruginosa , highlighting the necessity to select housekeeping genes according to the type of antibiotics used in the initial stage of experiment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Meng, Cao, Li, Li, Xie, Shi, Han, Shen and Liu.)

Syntrichia caninervis is the dominant bryophyte of the biological soil crusts found in the Gurbantunggut desert. The extreme desert environment is characterized by prolonged drought, temperature extremes, high radiation and frequent cycles of hydration and dehydration. S. caninervis is an ideal organism for the identification and characterization of genes related to abiotic stress tolerance. RT-qPCR expression analysis is a powerful analytical technique that requires the use of stable reference genes. Using available S. caninervis transcriptome data, we selected 15 candidate reference genes and analyzed their relative expression stabilities in S. caninervis gametophores exposed to a range of abiotic stresses or a hydration-desiccation-rehydration cycle. The programs geNorm, NormFinder, and RefFinder were used to assess and rank the expression stability of the 15 candidate genes. The stability ranking results of reference genes under each specific experimental condition showed high consistency using different algorithms. For abiotic stress treatments, the combination of two genes (α-TUB2 and CDPK) were sufficient for accurate normalization. For the hydration-desiccation-rehydration process, the combination of two genes (α-TUB1 and CDPK) were sufficient for accurate normalization. 18S was among the least stable genes in all of the experimental sets and was unsuitable as reference gene in S. caninervis. This is the first systematic investigation and comparison of reference gene selection for RT-qPCR work in S. caninervis. This research will facilitate gene expression studies in S. caninervis, related moss species from the Syntrichia complex and other mosses.

The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga. [ABSTRACT FROM AUTHOR]

International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species. [ABSTRACT FROM AUTHOR]