Evaluation of Reference Genes for Transcriptional Profiling in Two Cockroach Models.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhu S
PROVIDER: S-EPMC8701133 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Background:Due to cowpea ability to fix nitrogen in poor soils and relative tolerance to drought and salt stresses, efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Real-time quantitative PCR (qPCR) has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. In the present study, nine candidate reference genes were rigorously tested for their application in normalization of qPCR data onto roots of four distinct cowpea accessions under two abiotic stresses: root dehydration and salt (NaCl, 100 mM). In addition, the regulation of four target transcripts, under the same referred conditions was also scrutinized. Results:geNorm, NormFinder, BestKeeper, and ?Ct method results indicated a set of three statistically validated RGs for each stress condition: (I) root dehydration (actin, ubiquitin-conjugating enzyme E2 variant 1D, and a Phaseolus vulgaris unknown gene-UNK), and (II) salt (ubiquitin-conjugating enzyme E2 variant 1D, F-box protein, and UNK). The expression profile of the target transcripts suggests that flavonoids are important players in the cowpea response to the abiotic stresses analyzed, since chalcone isomerase and chalcone synthase were up-regulated in the tolerant and sensitive accessions. A lipid transfer protein also participates in the cowpea tolerance mechanisms to root dehydration and salt stress. The referred transcript was up-regulated in the two tolerant accessions and presented no differential expression in the sensitive counterparts. Chitinase B, in turn, generally related to plant defense, was an important target transcript under salt stress, being up-regulated at the tolerant, and down-regulated in the sensitive accession. Conclusions:Reference genes suitable for qPCR analyses in cowpea under root dehydration and salt stress were identified. This action will lead to a more accurate and reliable analysis of gene expression on this species. Additionally, the results obtained in this study may guide future research on gene expression in cowpea under other abiotic stress types that impose osmotic imbalance. The target genes analyzed, in turn, deserve functional evaluation due to their transcriptional regulation under stresses and biotechnological potential.
| S-EPMC6182843 | biostudies-literature
Project description:The data here described pertain to the article by Pojo et al. (2015) [10] titled “A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide” (Pojoet al., 2015 [10]). HOX genes are part of the homeobox gene family, which encodes transcription factors crucial during embryonic development (Grier et al., 2005; Pearson et al., 2005 [6,9]) and also in post developmental regulation(Neville et al., 2002; Yamamoto et al., 2003; Takahashi et al., 2004;Morgan 2006 [8,14,13,7]). Alterations interfering with the regulation of these genes may lead to tumorigenesis in adults. Due to their contributions in the control of important cellular processes, the deregulation of HOX genes is ultimately correlated with cancer treatment failure and patients' poor prognosis (Golub et al., 1999; Abdel-Fattah et al., 2006 [5,1]; Costa et al.,2010 [4]; Pojo et al., 2015 [10]). Recently, our studies showed that HOXA9 overexpression is associated with poor prognosis in patients with glioblastoma (GBM), the most common and most malignant primary brain tumor. Mechanistically, HOXA9 is associated with resistance to chemotherapy and with pro-proliferative, pro invasive and anti-apoptotic features (Costa et al., 2010 [4]; Pojo et al., 2015 [10]) in GBM in vitro models. Since HOXA9 is a transcription factor, its target genes can be the true biological effectors of its aggressiveness. In this context, whole genome Agilent's microarrays were used to obtain the full transcriptome of HOXA9 in a variety of GBM cell models, including human immortalized astrocytes, established GBM cell lines, and GBM patient derived cell cultures. Here, we provide detailed methods, including experimental design and microarray data analyses,which can be accessed in Gene Expression Omnibus (GEO) under the accession number GSE56517. Additional interpretation of the data is included and supplemented in (Pojo et al., 2015 [10]).
| S-EPMC4583997 | biostudies-literature
Project description:Carex muskingumensis is a highly valued perennial ornamental grass cultivated worldwide. However, there is limited genetic data regarding this species. Selection of proper reference genes (RGs) for reverse transcription quantitative PCR (RT-qPCR) data normalization has become an essential step in gene expression analysis. In this study, we aimed to examine expression stability of nine candidate RGs in C. muskingumensis plants, subjected to osmotic stress, generated either by salinity or PEG treatment. The identification of genes exhibiting high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt method). The results showed that the combination of two genes would be sufficient for reliable expression data normalization. ADP (ADP-ribosylation factor) and TBP (TATA-box-binding protein) were identified as the most stably expressed under salinity treatment, while eIF4A (eukaryotic initiation factor 4A) and TBP were found to show the highest stability under PEG-induced drought. A set of three genes (ADP, eIF4A and TBP) displayed the highest expression stability across all experimental samples tested in this study. To our best knowledge, this is the first report regarding RGs selection in C. muskingumensis. It will provide valuable starting point information for conducting further analyses in this and related species concerning their responses to water shortage and salinity stress.
| S-EPMC7563777 | biostudies-literature
Project description:Prion infection results in progressive neurodegeneration of the central nervous system invariably resulting in death. The pathological effects of prion diseases in the brain are morphologically well defined, such as gliosis, vacuolation, and the accumulation of disease-specific protease-resistant prion protein (PrPSc). However, the underlying molecular events that lead to the death of neurons are poorly characterised.In this study cDNA microarrays were used to profile gene expression changes in the brains of two different strains of mice infected with three strains of mouse-adapted scrapie. Extensive data was collected and analyzed, from which we identified a core group of 349 prion-related genes (PRGs) that consistently showed altered expression in mouse models. Gene ontology analysis assigned many of the up-regulated genes to functional groups associated with one of the primary neuropathological features of prion diseases, astrocytosis and gliosis; protein synthesis, inflammation, cell proliferation and lipid metabolism. Using a computational tool, Ingenuity Pathway Analysis (IPA), we were able to build networks of interacting genes from the PRG list. The regulatory cytokine TGFB1, involved in modulating the inflammatory response, was identified as the outstanding interaction partner for many of the PRGs. The majority of genes expressed in neurons were down-regulated; a number of these were involved in regulatory pathways including synapse function, calcium signalling, long-term potentiation and ERK/MAPK signalling. Two down-regulated genes coding for the transcription regulators, EGR1 and CREB1, were also identified as central to interacting networks of genes; these factors are often used as markers of neuronal activity and their deregulation could be key to loss of neuronal function.These data provides a comprehensive list of genes that are consistently differentially expressed in multiple scrapie infected mouse models. Building networks of interactions between these genes provides a means to understand the complex interplay in the brain during neurodegeneration. Resolving the key regulatory and signaling events that underlie prion pathogenesis will provide targets for the design of novel therapies and the elucidation of biomarkers.
| S-EPMC2294129 | biostudies-literature
Project description:Disease modeling requires appropriate cellular models that best mimic the underlying pathophysiology. Human origin and an adequate expression of the disease protein are pre-requisites that support information from a model to be meaningful. In this study we investigated expression profiles of (i) PBMCs and (ii) fibroblasts as patient derived cells as well as (iii) lymphoblasts and (iv) induced pluripotent stem cells (iPSC) as immortalized sources, and (v) iPSC-derived cortical neurons to assess their aptitude to model motor neuron diseases (MNDs) including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). We generated all five different cell types from two healthy donors and performed RNA sequencing to display expression patterns in MND-related genes. For the ten most common HSP genotypes we validated gene expression by qPCR. To verify the results on protein level, proteome analysis of fibroblasts, iPSCs and cortical neurons was performed. Depending on the specific MND gene we found largely different expression patterns. Out of 168 MND-related genes, 50 had their highest expression in iPSC-derived cortical neurons, 41 were most strongly expressed in fibroblasts, 26 in lymphoblasts, 22 in iPSCs, and 14 in PBMCs. Pathophysiologically related MNDs like HSPs associated with axonal transport deficits shared highest expression in cortical neurons. 15 MND-related genes were not detectable in any of the analyzed cell types. This may reflect the critical dependency of motor neurons on support of other cell types like oligodendrocytes which express myelin proteins like L1CAM (SPG1), PLP1 (SPG2) and MAG (SPG75) which are lacking in neurons but cause MNDs if mutated. This study provides comprehensive information on expression of genes associated with a large spectrum of MNDs. Expression profiles can be used to inform on appropriate cell models for genotype specific motor neuron research.
| S-EPMC7543453 | biostudies-literature
Project description:The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.
| S-EPMC8613261 | biostudies-literature
Project description:BackgroundQuantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori.MethodsWe evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes.ResultsWe show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards.ConclusionsThis work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.
| S-EPMC4417234 | biostudies-literature
Project description:BackgroundQuantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa "Nisqually 1" and P. tremula x P. alba 717 1-B4) following MIQE guidelines.ResultsWe evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNorm(PLUS) and BestKeeper. GeNorm(PLUS) ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes.ConclusionsThis is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.
| S-EPMC3479007 | biostudies-literature
Project description:The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic, receptor-ligand interactions and adaptive-like responses to viral infections. In the present study, we generated a single-cell transcriptional reference map of healthy human blood- and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene-regulatory networks defining NK cell differentiation. Transfer learning facilitated incorporation of tumor-infiltrating NK cell transcriptomes (39 datasets, 7 solid tumors, 427 patients) into the reference map to analyze tumor microenvironment (TME)-induced perturbations. Of the six functionally distinct NK cell states identified, a dysfunctional stressed CD56bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant effector CD56dim state were commonly enriched across tumor types, the ratio of which was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of new NK cell therapies and can be extended through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.
| S-EPMC11291284 | biostudies-literature
Project description:Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.
| S-EPMC4361542 | biostudies-literature