Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.
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ABSTRACT:
SUBMITTER: Chen C
PROVIDER: S-EPMC4822748 | biostudies-literature | 2016 Jan-Mar
REPOSITORIES: biostudies-literature
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Project description:The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis-an obligate aphid pathogenic fungus-the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.
| S-EPMC5495205 | biostudies-literature
Project description:Macrophomina phaseolina is a soil-borne pathogenic fungus that infects a wide range of crop species and causes severe yield losses. Although the genome of the fungus has been sequenced, the molecular basis of its virulence has not been determined. Identification of up-regulated genes during fungal infection is important to understand the mechanism involved in its virulence. To ensure reliable quantification, expression of target genes needs to be normalized on the basis of certain reference genes. However, in the case of M. phaseolina, reference genes or their expression analysis have not been reported in the literature. Therefore, the objective of this study was to evaluate 12 candidate reference genes for the expression analysis of M. phaseolina genes by applying three different fungal growth conditions: a) during root and stem infection of soybean, b) in culture media with and without soybean leaf infusion and c) by inoculating a cut-stem. Based on BestKeeper, geNorm and NormFinder algorithms, CYP1 was identified as the best recommended reference gene followed by EF1β for expression analysis of fungal gene during soybean root infection. Besides Mp08158, CYP1 gene was found suitable when M. phaseolina was grown in potato-dextrose broth with leaf infusion. In the case of cut-stem inoculation, Mp08158 and Mp11185 genes were found to be most stable. To validate the selected reference genes, expression analysis of two cutinase genes was performed. In general, the expression patterns were similar when the target genes were normalized against most or least stable gene. However, in some cases different expression pattern can be obtained when least stable gene is used for normalization. We believe that the reference genes identified and validated in this study will be useful for gene expression analysis during host infection with M. phaseolina.
| S-EPMC9355225 | biostudies-literature
Project description:Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most effective and sensitive techniques in gene expression assay, for which selection of reference genes is a prerequisite. In teleost species, such as Chinese perch, the expression profiling of miRNAs as reference genes for RT-qPCR has not been intensively studied. In the present study, the expression profiles of six miRNAs (miR-101a, miR-146a, miR-22a, miR-23a, miR-26a and let-7a) and one small nuclear RNA (U6) were assayed with RT-qPCR in different adult tissues, developmental stages and growth conditions of Chinese perch, Siniperca chuatsi. The analyses revealed that embryonic developmental stage is an important variability factor in the expression stability of miRNAs. All six miRNAs exhibited better expression consistency than U6 in most of the conditions examined, and therefore, they may be more suitable as a reference gene for miRNA quantification. When different tissues and developmental stages were considered, miR-22a demonstrated the most consistent expression pattern, and the best combination of reference genes was miR-22a and miR-23a. Our study offers useful data for selecting miRNAs as reference genes for RT-qPCR analysis of miRNAs in teleost fishes under different conditions.
| S-EPMC4425082 | biostudies-literature
Project description:Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), β-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), β-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.
| S-EPMC9769637 | biostudies-literature
Project description:We developed a R-based script to select internal control genes based solely on read counts and gene sizes. We used this method to pick custom reference genes for the differential expression analysis of three transcriptome sets from transgenic Arabidopsis plants expressing heterologous fungal effector proteins tagged with GFP (using GFP alone as the control). The custom reference genes showed lower covariance and fold change as well as a broader range of expression levels than commonly used reference genes. When analyzed with NormFinder, both typical and custom reference genes were considered suitable internal controls, but the custom selected genes were more stable. geNorm produced a similar result in which most custom selected genes ranked higher (i.e. were more stable) than commonly used reference genes.
2019-08-21 | GSE136038 | GEO
Project description:Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.
| S-EPMC7016721 | biostudies-literature
Project description:Huperzine A (HupA) is a powerful and selective inhibitor of acetylcholinesterase. It has attracted widespread attention endangering the ultimate plant sources of Lycopodiaceae family. In this study, we used Huperzia serrata, extensively used in Traditional Chinese medicine (TCM), a slow growing vascular plant as the model plant of the Lycopodiaceae family to develop and validate the reference genes. We aim to use gene expression platform to understand the gene expression of different tissues and developmental stages of this medicinal herb. Eight candidate reference genes were selected based on RNA-seq data and evaluated with qRT-PCR. The expression of L/ODC and cytochrome P450s genes known for their involvement in lycopodium alkaloid biosynthesis, were also studied to validate the selected reference genes. The most stable genes were TBP, GAPDH, and their combination (TBP + GAPDH). We report for the first time the reference gene of H. serrata's different tissues which would provide important insights into understanding their biological functions comparing other Lycopodiaceae plants and facilitate a good biopharming approach.
| S-EPMC6367274 | biostudies-literature
Project description:The rice blast fungus causes significant annual harvest losses. It also serves as a genetically-tractable model to study fungal ingress. Whilst pathogenicity determinants have been unmasked and changes in global gene expression described, we know little about Magnaporthe oryzae cell wall remodelling. Our interests, in wall remodelling genes expressed during infection, vegetative growth and under exogenous wall stress, demand robust choice of reference genes for quantitative Real Time-PCR (qRT-PCR) data normalisation. We describe the expression stability of nine candidate reference genes profiled by qRT-PCR with cDNAs derived during asexual germling development, from sexual stage perithecia and from vegetative mycelium grown under various exogenous stressors. Our Minimum Information for Publication of qRT-PCR Experiments (MIQE) compliant analysis reveals a set of robust reference genes used to track changes in the expression of the cell wall remodelling gene MGG_Crh2 (MGG_00592). We ranked nine candidate reference genes by their expression stability (M) and report the best gene combination needed for reliable gene expression normalisation, when assayed in three tissue groups (Infective, Vegetative, and Global) frequently used in M. oryzae expression studies. We found that MGG_Actin (MGG_03982) and the 40S 27a ribosomal subunit MGG_40s (MGG_02872) proved to be robust reference genes for the Infection group and MGG_40s and MGG_Ef1 (Elongation Factor1-?) for both Vegetative and Global groups. Using the above validated reference genes, M. oryzae MGG_Crh2 expression was found to be significantly (p<0.05) elevated three-fold during vegetative growth as compared with dormant spores and two fold higher under cell wall stress (Congo Red) compared to growth under optimal conditions. We recommend the combinatorial use of two reference genes, belonging to the cytoskeleton and ribosomal synthesis functional groups, MGG_Actin, MGG_40s, MGG_S8 (Ribosomal subunit 40S S8) or MGG_Ef1, which demonstrated low M values across heterogeneous tissues. By contrast, metabolic pathway genes MGG_Fad (FAD binding domain-containing protein) and MGG_Gapdh (Glyceraldehyde-3-phosphate dehydrogenase) performed poorly, due to their lack of expression stability across samples.
| S-EPMC4999194 | biostudies-literature
Project description:Background:Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a rapid and sensitive approach to identify miRNA and protein-coding gene expression in plants. However, because of the specially designated reverse transcription and shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA expression in plants, and different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of quantitative PCR of miRNA and mRNA. Results:In this study, a modified reverse transcription PCR protocol was adopted for selecting and validating universal internal reference genes of mRNAs and miRNAs. Eight commonly used reference genes, four stably expressed novel genes in Populus tremula, three small noncoding RNAs and three conserved miRNAs were selected as candidate genes, and the stability of their expression was examined across a set of 38 tissue samples from four developmental stages of poplar clone 84K (Populus alba?×?Populus glandulosa). The expression stability of these candidate genes was evaluated systematically by four algorithms: geNorm, NormFinder, Bestkeeper and DeltaCt. The results showed that Eukaryotic initiation factor 4A III (EIF4A) and U6-2 were suitable for samples of the callus stage; U6-1 and U6-2 were best for the seedling stage; Protein phosphatase 2A-2 (PP2A-2) and U6-1 were best for the plant stage; and Protein phosphatase 2A-2 (PP2A-2) and Oligouridylate binding protein 1B (UBP) were the best reference genes in the adventitious root (AR) regeneration stage. Conclusions:The purpose of this study was to identify the most appropriate reference genes for qRT-PCR of miRNAs and mRNAs in different tissues at several developmental stages in poplar. U6-1, EIF4A and PP2A-2 were the three most appropriate reference genes for qRT-PCR normalization of miRNAs and mRNAs during the plant regeneration process, and PP2A-2 and UBP represent the best reference genes in the AR regeneration stage of poplar. This work will benefit future studies of expression and function analysis of miRNAs and their target genes in poplar.
| S-EPMC6451301 | biostudies-literature
Project description:BACKGROUND: Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. FINDINGS: Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. CONCLUSION: Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.
| S-EPMC3854615 | biostudies-literature