An Ignored Pitfall in Selecting Reference Genes and the Solution with Emphasis on Subtypes and Sex of Thyroid Cancer Patients

2021 ◽  
Author(s):  
Ghazal Esfandiarpour ◽  
Mohammad Mokhtari ◽  
seyed-Morteza Javadirad ◽  
Mohsen Kolahdouzan ◽  
Ahmed Almuslimawi
Keyword(s):  
Thyroid Cancer ◽  
Statistical Analysis ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Meta Analysis ◽  
Specific Reference ◽  
Cancer Subtypes ◽  
Common Reference ◽  
Selection Of

Abstract Routine tissue specific reference genes are widely used in transcriptomics studies without concerning their target genes, sex of patients, and diseases subtype. We proposed the concept of specific reference genes for each target gene after considering sex of patients and thyroid cancer subtypes. RT-qPCR technique was coupled with expression meta-analysis of samples with different races and ethnicities, in both sexes, and in different thyroid cancer subtypes. Eight common reference genes were evaluated and some of them undoubtedly ruled out. We found that mean and SD values of the genes must be considered carefully before the selection of reference genes. A formula was also developed accordingly and we equipped it with statistical analysis of more than 25000 genes. In conclusion, the reckless selection of reference genes can distort the output and must be prohibited.

Copper affects the binding of HIF-1α to the critical motifs of its target genes

Metallomics ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 429-438 ◽  
Author(s):  
Zhijuan Wu ◽  
Wenjing Zhang ◽  
Y. James Kang
Keyword(s):  
Dna Binding ◽  
Target Gene ◽  
Target Genes ◽  
Gene Selection ◽  
Hypoxic Conditions ◽  
Selection Of

Copper regulates the target gene selection of HIF-1α under hypoxic conditions by affecting HIF-1α-DNA binding patterns across the genome.

scholarly journals Choosing reference genes for RT-qPCR for Fusarium graminearum plant infection (In Planta) and In Vitro growth studies based on transcriptomic data

2019 ◽  
Author(s):  
Xiaorong Lin ◽  
Hongchen Li ◽  
Zonghua Wang ◽  
Stefan Olsson
Keyword(s):  
Fusarium Graminearum ◽  
Reference Genes ◽  
Target Genes ◽  
Housekeeping Genes ◽  
Relative Difference ◽  
Transcriptome Data ◽  
In Planta ◽  
Plant Infection ◽  
Selection Of

Background. Choosing reference genes for RT-qPCR for the study of transcriptomic responses of target genes is often done using “standard” reference genes (housekeeping genes) selected before the genomic era. Now, published transcriptome data can be used to aid in this selection to avoid the selection of a reference gene that varies and obscure results. Methods. We use transcriptome data for the model pathogen fungus Fusarium graminearum to select housekeeping genes for In Vitro and In Planta conditions. Transcriptome data was downloaded from a publicly available database. We selected a database where transcriptome chip data from many experiments using the same chip has been deposited divided the downloaded data into In Vitro and In Planta conditions based on the information about the experiments. Results. We ranked the genes with the least variation (relative difference between maximum and minimum expression) across each dataset. Genes previously shown to perform well as reference genes for In Vitro conditions in a similar analysis as ours also performed well for In Vitro conditions in our dataset but worked less well for In Planta conditions. We found 5 reference genes that performed well under both In Planta conditions and In Vitro conditions. Discussion. Even if these 5 reference genes performed well, for other (new) target conditions we recommend making a transcriptome analysis to select well performing reference genes for RT-qPCR if possible. Alternatively, select 2 of the 5 genes that we show here performed well under both In Planta and In Vitro conditions.

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 LINE-1 Endonuclease-Dependent Retrotranspositional Events Causing Human Genetic Disease: Mutation Detection Bias and Multiple Mechanisms of Target Gene Disruption

2006 ◽  
Vol 2006 ◽  
pp. 1-9 ◽  
Author(s):  
Jian-Min Chen ◽  
Claude Férec ◽  
David N. Cooper
Keyword(s):  
Genetic Disease ◽  
Gene Disruption ◽  
Target Gene ◽  
Target Genes ◽  
Mutation Detection ◽  
Meta Analysis ◽  
Human Genetic Disease ◽  
Detection Bias ◽  
Target Gene Disruption ◽  
Key Issues

LINE-1 (L1) elements are the most abundant autonomous non-LTR retrotransposons in the human genome. Having recently performed a meta-analysis of L1 endonuclease-mediated retrotranspositional events causing human genetic disease, we have extended this study by focusing on two key issues, namely, mutation detection bias and the multiplicity of mechanisms of target gene disruption. Our analysis suggests that whereas an ascertainment bias may have generally militated against the detection of autosomal L1-mediated insertions, autosomal L1 direct insertions could have been disproportionately overlooked owing to their unusually large size. Our analysis has also indicated that the mechanisms underlying the functional disruption of target genes by L1-mediated retrotranspositional events are likely to be dependent on several different factors such as the type of insertion (L1 direct, L1trans-drivenAlu, or SVA), the precise locations of the inserted sequences within the target gene regions, the length of the inserted sequences, and possibly also their orientation.

scholarly journals Long non-coding RNA LINC00488 facilitates thyroid cancer cell progression through miR-376a-3p/PON2

2021 ◽  
Vol 41 (3) ◽  
Author(s):  
Fuyuan Xie ◽  
Longgen Li ◽  
Yuting Luo ◽  
Rensheng Chen ◽  
Jinhong Mei
Keyword(s):  
Thyroid Cancer ◽  
Cancer Cell ◽  
Target Gene ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Thyroid Cancer Cell ◽  
Gene Assay

Abstract Objective: Long non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. The aim of the present study was to identify a functional lncRNA LINC00488 and its effects on thyroid cancer in the view of cell proliferation and apoptosis. Methods: In order to evaluate the effects of LINC00488 on the cellular process of thyroid cancer, we performed a series of in vitro experiments, including cell counting kit-8 (CCK-8) assay, EdU (5-ethynyl-2′-deoxyuridine) assay, flow cytometry, transwell chamber assay, Western blot and RT-qPCR. The target gene of LINC00488 was then identified by bioinformatics analysis (DIANA and TargetScan). Finally, a series of rescue experiments was conducted to validate the effect of LINC00488 and its target genes on proliferation, migration, invasion and apoptosis of thyroid cancer. Results: Our findings revealed that LINC00488 was highly expressed in thyroid cancer cell lines (BCPAP, BHP5-16, TPC-1 and CGTH-W3) and promoted the proliferation, migration and invasion, while inhibited the apoptosis of thyroid cancer cells (BCPAP and TPC-1). The results of bioinformatics analysis and dual luciferase reporter gene assay showed that LINC00488 could directly bind to miR-376a-3p and down-regulated the expression level of miR-376a-3p. In addition, Paraoxonase-2 (PON2) was a target gene of miR-376a-3p and negatively regulated by miR-376a-3p. Rescue experiment indicated that LINC00488 might enhance PON2 expression by sponging miR-376a-3p in thyroid cancer. Conclusion: Taken together, our study revealed that lncRNA LINC00488 acted as an oncogenic gene in the progression of thyroid cancer via regulating miR-376a-3p/PON2 axis, which indicated that LINC00488-miR-376a-3p-PON2 axis could serve as novel biomarkers or potential targets for the treatment of thyroid cancer.

scholarly journals Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1178
Author(s):  
Chang-Fei Guo ◽  
Hui-Peng Pan ◽  
Li-He Zhang ◽  
Da Ou ◽  
Zi-Tong Lu ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Reference Genes ◽  
Target Genes ◽  
Southern China ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Pairwise Variation ◽  
Specific Reference ◽  
Qpcr Analysis ◽  
Different Temperatures

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiata and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm, ΔCt, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage; PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen); EF1A and ArgK between two populations; β-tubulin and EF1A for different temperatures (5, 15, 25, 35 °C); and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.

scholarly journals Validation of reference genes for RT-qPCR in cardiac tissue of rats induced to obesity and diabetes

2020 ◽  
Vol 9 (11) ◽  
pp. e1599119702
Author(s):  
Marina Martins de Oliveira ◽  
Kalynka Gabriella do Livramento ◽  
Maísa Lamounier Magalhães ◽  
Luciano Vilela Paiva ◽  
Ana Paula Peconick
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Cardiac Tissue ◽  
Rna Extraction ◽  
Obesity And Diabetes ◽  
The Stability ◽  
Universal Reference

The validation of reference genes is an essential step for any RT-qPCR analysis. In this way, the present paper aimed to identify and validate reference genes for RT-qPCR in cardiac tissue of rats of the Rattus norvegicus albinus specie, submitted to obesity associated or not to type 2 diabetes mellitus. For this, the metabolic changes were induced at the 42nd day of life and the euthanasia was performed on the 70th day. The heart apexes were collected and destinates for RNA extraction. The RT-qPCR technique was performed in own thermocycler, the efficiency of the primers found by the LinReg software and the stability of the expression of the reference genes in the samples was analyzed by the RefFinder algorithm. The candidates for reference genes were GAPDH, POLR2A, RPL32, and RPL4 and the target gene used to verify the differences in gene expression of candidates for reference genes was CMA1. The obese animals showed a decrease in CMA1 gene expression when compared to the two most stable reference genes. The opposite occurs when it is compared to the two less stable reference genes. The GAPDH and POLR2A genes are the best to normalize the reactions with the samples in question. There is no universal reference gene for all situations, which requires systematic validation for each situation. The use of unvalidated reference genes may compromise the interpretation of the expression of the target genes, which would prevent the reflection of the actual situation.

scholarly journals EP300 and SIRT1/6 Co-Regulate Lapatinib Sensitivity Via Modulating FOXO3-Acetylation and Activity in Breast Cancer

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1067 ◽  
Author(s):  
Zimam Mahmud ◽  
Ana R. Gomes ◽  
Hee Jin Lee ◽  
Sathid Aimjongjun ◽  
Yannasittha Jiramongkol ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Target Gene ◽  
Target Genes ◽  
Ectopic Expression ◽  
Sirtuin 1 ◽  
Fine Tuning ◽  
Gene Promoters ◽  
Breast Cancers ◽  
Her2 Positive ◽  
Cancer Subtypes

Forkhead Box O3 (FOXO3) is a tumor suppressor whose activity is fine-tuned by post-translational modifications (PTMs). In this study, using the BT474 breast cancer cells and a recently established lapatinib resistant (BT474-LapR) cell line, we observed that higher FOXO3 and acetylated (Ac)-FOXO3 levels correlate with lapatinib sensitivity. Subsequent ectopic expression of EP300 led to an increase in acetylated-FOXO3 in sensitive but not in resistant cells. Drug sensitivity assays revealed that sensitive BT474 cells show increased lapatinib cytotoxicity upon over-expression of wild-type but not acetylation-deficient EP300. Moreover, FOXO3 recruitment to target gene promoters is associated with target gene expression and drug response in sensitive cells and the inability of FOXO3 to bind its target genes correlates with lapatinib-resistance in BT474-LapR cells. In addition, using SIRT1/6 specific siRNAs and chemical inhibitor, we also found that sirtuin 1 and -6 (SIRT1 and -6) play a part in fine-tuning FOXO3 acetylation and lapatinib sensitivity. Consistent with this, immunohistochemistry results from different breast cancer subtypes showed that high SIRT6/1 levels are associated with constitutive high FOXO3 expression which is related to FOXO3 deregulation/inactivation and poor prognosis in breast cancer patient samples. Collectively, our results suggest the involvement of FOXO3 acetylation in regulating lapatinib sensitivity of HER2-positive breast cancers.

scholarly journals Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7319 ◽  
Author(s):  
Guanglong Wang ◽  
Chang Tian ◽  
Yunpeng Wang ◽  
Faxiang Wan ◽  
Laibao Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Reference Genes ◽  
Expression Profiles ◽  
Accurate Determination ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Specific Reference ◽  
Variable Expression ◽  
Selection Of

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

scholarly journals Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage

2020 ◽  
Vol 11 ◽  
Author(s):  
Liang Liu ◽  
Hui Han ◽  
Qingxian Li ◽  
Ming Chen ◽  
Siqi Zhou ◽  
...  
Keyword(s):  
Reference Genes ◽  
Target Genes ◽  
Male Rat ◽  
Fluorescence Quantitative Pcr ◽  
Genes Expression ◽  
Before And After ◽  
Developing Rat ◽  
Detect Gene Expression ◽  
Gene Expression Levels ◽  
Selection Of

Real-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT-qPCR is conducive to obtaining accurate experimental results. However, the selection of the panel of reference genes for RT-qPCR in rat developing cartilage has not been well documented. In this study, we selected eight reference genes commonly used in rat cartilage from literature (GAPDH, ACTB, 18S, GUSB, HPRT1, RPL4, RPL5, and SDHA) as candidates. Then, we screened out the optimal panel of reference genes in female and male rat cartilage of fetus (GD20), juvenile (PW6), and puberty (PW12) in physiology with stability analysis software of genes expression. Finally, we verified the reliability of the selected panel of reference genes with the rat model of intrauterine growth retardation (IUGR) induced by prenatal dexamethasone exposure (PDE). The results showed that the optimal panel of reference genes in cartilage at GD20, PW6, and PW12 in physiology was RPL4 + RPL5, which was consistent with the IUGR model, and there was no significant gender difference. Further, the results of standardizing the target genes showed that RPL4 + RPL5 performed smaller intragroup differences than other panels of reference genes or single reference genes. In conclusion, we found that the optimal panel of reference genes in female and male rat developing cartilage was RPL4 + RPL5, and there was no noticeable difference before and after birth.

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