scholarly journals Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Aiqin Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Transcriptional Level ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Qrt Pcr

Abstract Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

scholarly journals Validation of internal reference genes for relative quantitation studies of gene expression in human laryngeal cancer

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2763 ◽  
Author(s):  
Xiaofeng Wang ◽  
Jinting He ◽  
Wei Wang ◽  
Ming Ren ◽  
Sujie Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Laryngeal Cancer ◽  
Reference Genes ◽  
Target Gene ◽  
Cancer Tissue ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Internal Reference Gene ◽  
Relative Quantitation

BackgroundThe aim of this study was to determine the expression stabilities of 12 common internal reference genes for the relative quantitation analysis of target gene expression performed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in human laryngeal cancer.MethodsHep-2 cells and 14 laryngeal cancer tissue samples were investigated. The expression characteristics of 12 internal reference gene candidates (18S rRNA, GAPDH, ACTB, HPRT1, RPL29, HMBS, PPIA, ALAS1, TBP, PUM1, GUSB, and B2M) were assessed by RT-qPCR. The data were analyzed by three commonly used software programs: geNorm, NormFinder, and BestKeeper.ResultsThe use of the combination of four internal reference genes was more appropriate than the use of a single internal reference gene. The optimal combination was PPIA + GUSB + RPL29 + HPRT1 for both the cell line and tissues; while the most appropriate combination was GUSB + RPL29 + HPRT1 + HMBS for the tissues.ConclusionsOur recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on laryngeal tumors.

scholarly journals Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6834
Author(s):  
Teizhu Yang ◽  
Bingning Gu ◽  
Guolyu Xu ◽  
Yanmei Shi ◽  
Heding Shen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Osmotic Pressure ◽  
Reference Gene ◽  
Salinity Stress ◽  
Reference Genes ◽  
Target Gene ◽  
Housekeeping Genes ◽  
Target Gene Expression ◽  
Qrt Pcr ◽  
Suitable Reference

Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions.

scholarly journals Evaluation and Validation of Reference Genes for Quantitative Real-Time PCR in Helopeltis theivora Waterhouse (Hemiptera: Miridae)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Shengfeng Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Transcriptional Level ◽  
Quantitative Real Time Pcr ◽  
Systematic Analysis ◽  
Helopeltis Theivora ◽  
Qrt Pcr

Abstract Helopeltis theivora Waterhouse is a predominant sucking pest in many tropic economic crops, such as tea, cocoa and coffee. Quantitative real-time PCR (qRT-PCR) is one of the most powerful tools to analyze the gene expression level and investigate the mechanism of insect physiology at transcriptional level. Gene expression studies utilizing qRT-PCR have been applied to numerous insects so far. However, no universal reference genes could be used for H. theivora. To obtain accurate and reliable normalized data in H. theivora, twelve candidate reference genes were examined under different tissues, developmental stages and sexes by using geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder algorithms, respectively. The results revealed that the ideal reference genes differed across the treatments, and the consensus rankings generated from stability values provided by these programs suggested a combination of two genes for normalization. To be specific, RPS3A and Actin were the best suitable reference genes for tissues, RPL13A and GAPDH were suitable for developmental stages, EF1α and RPL13A were suitable for sexes, and RPL13A and RPS3A were suitable for all samples. This study represents the first systematic analysis of reference genes for qRT-PCR experiments in H. theivora, and the results can provide a credible normalization for qRT-PCR data, facilitating transcript profiling studies of functional genes in this insect.

scholarly journals Selection of Reference Genes for Normalization of Real-Time PCR Data in Calliptamus italicus (Orthoptera: Acrididae) Under Different Temperature Conditions

2019 ◽  
Vol 19 (6) ◽  
Author(s):  
Hongxia Hu ◽  
Xiaofang Ye ◽  
Han Wang ◽  
Rong Ji
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Target Gene Expression

Abstract Global warming has dominated worldwide climate change trends, and adaptability to high temperatures is the main factor underlying the spread of the pest Calliptamus italicus in Xinjiang Province, China. However, knowledge about the molecular mechanisms responsible for this adaptability and other related biological properties of C. italicus remain relatively unclear. Real-time quantitative polymerase chain reaction (RT-qPCR) is a key tool for gene expression analysis associated with various biological processes. Reference genes are necessary for normalizing gene expression levels across samples taken from specific experimental conditions. In this study, transcript level of five genes (GAPDH, 18S, TUB, ACT, and EF1α), commonly used as reference genes, were evaluated under nine different temperatures (27, 30, 33, 36, 39, 42, 45, 48, and 51°C) to assess their expression stability and further select the most suitable to be used on normalization of target gene expression data. Gene expression profiles were analyzed using geNorm, NormFinder, and BestKeeper software packages. The combined results demonstrated that the best-ranked reference genes for C. italicus are EF1α, GAPDH, and ACT under different thermal stress conditions. This is the first study that assesses gene expression analysis across a range of temperatures to select the most appropriate reference genes for RT-qPCR data normalization in C. italicus. These results should assist target gene expression analysis associated with heat stress in C. italicus.

scholarly journals On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

2021 ◽  
Author(s):  
Philipp Moritz Fricke ◽  
Angelika Klemm ◽  
Michael Bott ◽  
Tino Polen
Keyword(s):  
Gene Expression ◽  
Acetic Acid ◽  
Literature Search ◽  
Target Gene ◽  
Target Genes ◽  
Recombinant Dna ◽  
Acetic Acid Bacteria ◽  
Homoserine Lactone ◽  
Expression Systems ◽  
Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

scholarly journals Identification and validation of miRNA reference genes in poplar under pathogen stress

2021 ◽  
Author(s):  
Lichun Zhang ◽  
Xiaoqian Yang ◽  
Yiyi Yin ◽  
Jinxing Wang ◽  
Yanwei Wang
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Small Rna ◽  
Reference Genes ◽  
Expression Profiles ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Internal Standards ◽  
Qrt Pcr ◽  
Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

scholarly journals Effect of miR-27b-5p on apoptosis of human vascular endothelial cells induced by simulated microgravity

APOPTOSIS ◽  
2019 ◽  
Vol 25 (1-2) ◽  
pp. 73-91 ◽  
Author(s):  
Yi-Kai Pan ◽  
Cheng-Fei Li ◽  
Yuan Gao ◽  
Yong-Chun Wang ◽  
Xi-Qing Sun
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Target Gene ◽  
Simulated Microgravity ◽  
Target Genes ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Luciferase Assay ◽  
Qrt Pcr ◽  
Differentially Expressed Mirnas

AbstractWeightlessness-induced cardiovascular dysfunction can lead to physiological and pathological consequences. It has been shown that spaceflight or simulated microgravity can alter expression profiles of some microRNAs (miRNAs). Here, we attempt to identify the role of miRNAs in human umbilical vein endothelial cells (HUVECs) apoptosis under simulated microgravity. RNA-sequencing and quantitative real-time PCR (qRT-PCR) assays were used to identify differentially expressed miRNAs in HUVECs under simulated microgravity. Then we obtained the target genes of these miRNAs through target analysis software. Moreover, GO and KEGG enrichment analysis were performed. The effects of these miRNAs on HUVECs apoptosis were evaluated by flow cytometry, Western blot and Hoechst staining. Furthermore, we obtained the target gene of miR-27b-5p by luciferase assay, qRT-PCR and Western blot. Finally, we investigated the relationship between this target gene and miR-27b-5p in HUVECs apoptosis under normal gravity or simulated microgravity. We found 29 differentially expressed miRNAs in HUVECs under simulated microgravity. Of them, the expressions of 3 miRNAs were validated by qRT-PCR. We demonstrated that miR-27b-5p affected HUVECs apoptosis by inhibiting zinc fingers and homeoboxes 1 (ZHX1). Our results reported here demonstrate for the first time that simulated microgravity can alter the expression of some miRNAs in HUVECs and miR-27b-5p may protect HUVECs from apoptosis under simulated microgravity by targeting ZHX1.

The Interaction Between DOT1L and AF10 Is Required for H3K79 Dimethylation and MLL-AF9 Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 401-401
Author(s):  
Aniruddha J Deshpande ◽  
Liying Chen ◽  
Kathrin M Bernt ◽  
Stuart Dias ◽  
Deepti Banka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Target Gene ◽  
Leucine Zipper ◽  
Target Genes ◽  
Fusion Gene ◽  
Transformed Cells ◽  
Target Gene Expression ◽  
Transforming Activity ◽  
H3k79 Methylation

Abstract Abstract 401 MLL-fusion proteins induce changes in histone modifications that result in the abnormal and sustained expression of downstream oncogenic target genes. A number of recent studies have identified aberrant histone 3 lysine 79 (H3K79) methylation by the chromatin modifying enzyme DOT1L as an important epigenetic modification that sustains MLL-target gene expression. Aberrant H3K79 methylation has been shown to be necessary for oncogenic transformation mediated by a number of MLL-fusions. These recent findings have generated tremendous interest in H3K79 methylation as a therapeutic target in the MLL rearranged leukemias. The plant-homeodomain (PHD) and leucine zipper-containing protein AF10 biochemically interacts with DOT1L and is believed to influence H3K79 methylation. We generated conditional knockout mice in which the Dot1l-interacting octapeptide-motif leucine zipper (OM-LZ) domain of Af10 was flanked by LoxP sites. Deletion of the Af10OM-LZ domain with the Cre recombinase is predicted to abrogate the Af10-Dot1l interaction. Deletion of the Af10OM-LZ domain greatly reduced global H3K79 dimethylation as assessed by immunoblotting as well as mass spectrometry in Af10OM-LZ deleted HoxA9/Meis1a transformed cells. Given the importance of H3K79 methylation in MLL-rearranged leukemias, we sought to assess whether the transforming activity of the MLL-AF9 fusion gene was dependent on the Af10-Dot1l interaction. Using an MLL-AF9-IRES-GFP encoding retrovirus, we established immortalized blast-colony forming cultures from mouse lineage negative Sca-1 positive/Kit positive (LSK) bone marrow cells bearing floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain with Cre-recombinase dramatically reduced H3K79me2 on the MLL-target genes Hoxa5-10 and Meis1, leading to downregulation of these transcripts. We performed colony-forming cell (CFC) assays from MLL-AF9 transformed cells in the presence or absence of the Af10OM-LZ allele. In the first week, Af10OM-LZ deletion profoundly impaired the blast-colony forming potential of MLL-AF9 transformed LSKs and the only clones that could serially replate in subsequent passages had escaped Af10OM-LZ excision. Af10OM-LZ deleted colonies were very small and spread-out and showed morphological features of terminal myeloid differentiation. In contrast, HoxA9/Meis1 transformed LSK cells expanded normally in the absence of the Af10OM-LZ domain. These results demonstrate that the Af10OM-LZ, much like Dot1l, is critical for the in vitro transforming activity of the MLL-AF9 fusion gene, but does not non-specifically inhibit cellular proliferation. We then sought to investigate the potential role of the Af10OM-LZ domain in the in vivo leukemogenic activity of MLL-AF9. We generated primary MLL-AF9 leukemias from LSKs harboring floxed Af10OM-LZ alleles. Deletion of the Af10OM-LZ domain in cells explanted from the MLL-AF9 primary leukemias led to a significant increase in the disease latency in secondary recipient mice. Moreover, limiting dilution analysis of MLL-AF9 leukemias with or without the Af10OM-LZ domain demonstrated a >100 fold decrease in the frequency of leukemia initiating cells in the absence of the Af10OM-LZ domain. Microarray analysis showed that a vast majority of MLL-AF9 target genes were significantly downregulated in Af10OM-LZ deleted as compared to Af10OM-LZ wildtype MLL-AF9 leukemias. However, the Af10OM-LZ deleted cells could still eventually cause leukemia. This is intriguing given that Af10OM-LZ deletion, similar to Dot1l deletion, leads to a significant reduction in H3K79 dimethylation as well as MLL-target gene expression. A more detailed analysis of H3K79 methylation using mass spectrometry revealed that in contrast to H3K79 dimethylation, global levels of H3K79 mono-methylation were largely unchanged in Af10OM-LZ deleted cells. This suggests the residual MLL-AF9 target gene expression seen in Af10OM-LZ deleted cells is maintained by H3K79 monomethylation. Our results demonstrate a surprising role for Af10 in the conversion of H3K79 monomethylation to dimethylation and reveal the AF10-DOT1L interaction as an attractive therapeutic target in MLL-rearranged leukemias. Disclosures: Armstrong: Epizyme: Consultancy.

scholarly journals E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

2007 ◽  
Vol 27 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Cynthia Timmers ◽  
Nidhi Sharma ◽  
Rene Opavsky ◽  
Baidehi Maiti ◽  
Lizhao Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Control Of Gene Expression ◽  
Target Gene Expression ◽  
Rb Phosphorylation ◽  
P53 Target Genes

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

2016 ◽  
Vol 107 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Y. Tan ◽  
X.-R. Zhou ◽  
B.-P. Pang
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Functional Studies ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

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