Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions

Banana (Musa spp.) is severely damaged by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Biocontrol by inducing systemic resistance has been considered as one of the most important strategies to improve plant health. Very few studies have investigated appropriate reference gene selection for RT‐qPCR (quantitative real‐time polymerase chain reaction) analysis suitable for conditions of systemic activated resistance. In this study, we assessed over a time‐course the expression of seven candidate reference genes (EF1, TUB, ACT1, ACT2, L2, RPS2 and RAN) for Cavendish cultivar Brazilian (Musa spp. AAA) and dwarf banana cultivar Guangfen No. 1 (Musa spp. ABB) that were inoculated by Bacillus subtilis strain TR21 and Foc. We choose these plants because they are commonly planted in Southern China. Expression stability of the candidate genes was evaluated using various software packages (GeNorm, NormFinder and BestKeeper). L2 and TUB genes displayed maximum stability in Guangfen No. 1. In Brazilian, ACT1 and TUB were the most stable genes. To further validate the suitability of the reference genes identified in this study, the expression of pathogenesis‐related 1 (PR1) gene under TR21 and Foc strains Foc004/Foc009 treatments was also studied. Identified reference genes in this work that are most suitable for normalizing gene expression data in banana under Fusarium wilt resistance induction conditions will contribute to the understanding of disease resistance mechanisms induced by biocontrol strains in banana.     

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel,Anguilla anguilla

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.     

Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells

Intramuscular fat (IMF) is an important trait influencing meat quality, and intramuscular stromal-vascular cell (MSVC) differentiation is a key factor affecting IMF deposition. Quantitative real-time PCR (qPCR) is often used to screen the differentially expressed genes during differentiation of MSVCs, where proper reference genes are essential. In this study, we assessed 31 of previously reported reference genes for their expression suitability in porcine MSVCs derived form longissimus dorsi with qPCR. The expression stability of these genes was evaluated using NormFinder, geNorm and BestKeeper algorithms. NormFinder and geNorm uncovered ACTB, ALDOA and RPS18 as the most three stable genes. BestKeeper identified RPL13A, SSU72 and DAK as the most three stable genes. GAPDH was found to be the least stable gene by all of the three software packages, indicating it is not an appropriate reference gene in qPCR assay. These results might be helpful for further studies in pigs that explore the molecular mechanism underlying IMF deposition.     

Assessment of candidate reference genes for expression studies in <Emphasis Type="Italic">Vicia faba</Emphasis> L. by real-time quantitative PCR

Faba bean (Vicia faba L.) cultivation has declined in recent years due to several factors, including diseases and anti-nutritional compounds in the seeds. The introduction of disease resistance and the elimination of anti-nutritional factors in new varieties are important objectives in any breeding program for the species. Because of the faba bean’s huge genome, it is necessary to rely on synteny with related species in order to identify candidate genes responsible for the character under study. Quantification of expression level of candidate genes could help to validate them. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression level. Real-time quantitative PCR was used for evaluate the expression stability of 11 candidate reference genes. A wide set of samples, including different tissues, genotypes and several inoculations for the most important pathogens were employed. The expression stability of the candidate genes was analyzed using two different algorithms, geNorm and NormFinder, and results obtained from both algorithms were highly correlated for each experimental set. In all cases, either ACT1, CYP2 or ELF1A genes performed as the most stable genes in our experimental sets. They also represent part of the best combination of genes according to the geNorm and NormFinder algorithms. Our data showed the wide expression range of the selected genes, confirming that no single reference gene had a stable expression under these conditions in the faba bean. We recommend the use of ACT1, CYP2 and ELF1A as the most suitable reference genes to normalize gene expression for future studies in V. faba.     

Validation of reference genes in the feline endometrium

The aim of the study was to find the most stable reference genes from: ACTB, GAPDH, RPL30, CYC, RPL17, RPS7 and YWHAZ in the feline endometrium. Three free software packages, geNorm, NormFinder and BestKeeper were used. In geNorm analysis, the most stable gene was RPS7 (at a primer concentration 1000 nM) or YWHAZ (500 and 250 nM). According to NormFinder and BestKeeper, ACTB (at all examined primer concentrations) followed by RPS7 and CYC were the most stable genes. Based on geNorm results at least two genes from among RPS7, RPL30, ACTB or YWHAZ should be chosen for Real Time-PCR result normalization.     

Validation of housekeeping genes for gene expression studies in an ice alga <Emphasis Type="Italic">Chlamydomonas</Emphasis> during freezing acclimation

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.     

Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut.     

基于转录组测序的姬松茸镉胁迫下内参基因筛选

[目的] 筛选不同实验条件下,尤其是镉胁迫下的姬松茸内参基因,为研究姬松茸镉富集相关基因功能研究奠定基础。[方法] 根据不同浓度Cd（0、2、5 mg/L）胁迫下菌丝中转录组表达量数据,筛选出18个候选基因,设计了30对引物;根据引物扩增的特异性、扩增效率初步筛选出来自不同基因的5对引物;运用qRT-PCR检测了5个基因在不同浓度Cd胁迫下的菌丝样品、原基、菌柄和菌盖中的C_t值,利用geNorm、NormFinder和BestKeeper对这些基因的表达稳定性进行评价。[结果] SGT2和STK是菌丝阶段Cd胁迫下最稳定的2个候选内参基因,GAPDH是不同组织中最稳定的内参基因,SGT2、STK和GAPDH是所有实验条件下最稳定的3个内参基因。[结论] 在姬松茸镉胁迫条件下,本研究筛选出的SGT2和STK比常用的内参基因表现出更强的稳定性。     

Identification of suitable reference genes for expression studies in pomegranate under different biotic and abiotic stress conditions

Pomegranate (Punica granatum L.) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. Eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ?Ct, geNorm, BestKeeper, NormFinder, and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. For normalizing target gene expression under wilt, nematode, bacterial blight, and abiotic stress conditions both GAPDH and CYPFreference genes are adequate for qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

     

巴斯德毕赤酵母不同生长阶段内参基因的筛选与验证

【背景】巴斯德毕赤酵母(Komagataella phaffii)是一种甲基营养型酵母,近年来作为生产重组蛋白和构建生物合成途径的细胞工厂受到广泛关注。实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)是巴斯德毕赤酵母表达系统研究中一种快速、高效的基因表达水平检测技术,但需要进行归一化处理才能保证所得结果的可靠性。【目的】筛选并验证巴斯德毕赤酵母在不同生长阶段最稳定的内参基因用于精准归一化RT-qPCR的结果。【方法】通过转录组数据分析初步筛选出16个候选内参基因(rps8b、rpl35a、rpl10、eif5a、rpl19a、por1、rpl23b、0887、tif1、ole1、rpl14b、gs、sun、sdh2、trx1和ccp1)。通过RT-qPCR技术得到候选内参基因的C_t值,利用qBASE软件中的geNorm程序综合NormFinder算法评估内参基因的表达稳定性。【结果】通过geNorm分析得出精准归一化所需的最佳内参基因个数为2,最稳定的基因是rpl19a和tif1,NormFinder分析得到稳定性最高的内参基因为tif1。此外,利用甲酸脱氢酶编码基因fdh和乙醇脱氢酶甲醛脱氢酶双功能酶的编码基因afdh对候选内参基因进行验证。【结论】巴斯德毕赤酵母不同生长阶段的RT-qPCR进行精准归一化需要tif1和rpl19a这2个内参基因,为相关功能基因的表达定量提供了可靠的分析依据,补充了RT-qPCR分析中的内参基因,为巴斯德毕赤酵母不同生长阶段的基因表达调控及其应用研究提供了新的参考。     

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.     

Selection of reference genes for tissue/organ samples on day 3 fifth‐instar larvae in silkworm,Bombyx mori

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth‐instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori.     

Selection of suitable reference genes for quantitative real-time PCR in trabecular meshwork cells under oxidative stress

Oxidative stress-induced dysfunction in trabecular meshwork (TM) cells is considered a major alteration that can lead to glaucoma. Hydrogen peroxide (H₂O₂) is the most widely used agent for inducing oxidation in TM cells in vitro. Quantitative real-time PCR (qPCR) is an important method for studying alterations in gene expression, and suitable (i.e. invariant) reference genes must be defined to normalize expression levels. In this study, eight common reference genes, i.e. PRS18, ACTB, B2M, GAPDH, PPIA, HPRT1, YWHAZ, and TBP, were evaluated for use in studies of H₂O₂-induced dysfunction in TM cells. Three established algorithms, geNorm, NormFinder, and BestKeeper, were used to analyze the reference genes. ACTB expression was least affected by H₂O₂ treatment in TM cells, and the combination of PPIA and HPRT1 was the most suitable gene pair for normalization. GAPDH and TBP were the most unstable genes and accordingly should be avoided in experiments with TM cells. These results provide a foundation for analyses of the mechanisms underlying glaucoma, and emphasize the importance of selecting suitable reference genes for qPCR studies.