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.

Identification of reference genes for gene expression studies during seed germination and seedling establishment in Ricinus communis L.

2014 ◽  
Vol 24 (4) ◽  
pp. 341-352 ◽  
Author(s):  
Paulo R. Ribeiro ◽  
Bas J. W. Dekkers ◽  
Luzimar G. Fernandez ◽  
Renato D. de Castro ◽  
Wilco Ligterink ◽  
...  
Keyword(s):  
Gene Expression ◽  
Seed Germination ◽  
Reference Genes ◽  
Target Genes ◽  
Ricinus Communis ◽  
Quantitative Polymerase Chain Reaction ◽  
Optimal Combination ◽  
Expression Levels ◽  
Gene Expression Studies ◽  
Gene Expression Levels

AbstractReverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

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.

Assessment of AXL and mTOR genes expression in medullary thyroid carcinoma (MTC) cell line in relation with over expression of miR-144 and miR-34a

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Shaghayegh Pishkari ◽  
Razie Hadavi ◽  
Ameneh Koochaki ◽  
Javad Razaviyan ◽  
Mahdi Paryan ◽  
...  
Keyword(s):  
Thyroid Carcinoma ◽  
Cell Line ◽  
Medullary Thyroid Carcinoma ◽  
Target Genes ◽  
Critical Role ◽  
Luciferase Assay ◽  
Medullary Thyroid ◽  
Genes Expression ◽  
Before And After ◽  
New Strategy

Abstract Objectives The aim of the present study was to investigate the expression of AXL and mTOR genes and their targeting microRNAs (miRNAs) including miR-34a and miR-144 in Medullary Thyroid Carcinoma (MTC) cell line, TT, and determine the effect of these two miRNAs on their target genes to introduce new molecular markers or therapeutics. Methods The expression of miR-34a, miR-144, and their targets genes including AXL and mTOR was evaluated by quantitative Real-time PCR. Luciferase assay was performed to confirm the interaction between miRNAs and their target mRNAs. The expression level of AXL and mTOR was evaluated before and after miRNAs induction in TT cell line compared with Cos7 as control cells. Results The expression of AXL and mTOR were up-regulated significantly, while miR-34a and miR-144 were down-regulated in TT cell line compared to Cos7. After transduction, the overexpression of miR-34a and 144 caused down-regulation of both genes. Luciferase assay results showed that the mTOR is targeted by miR-34a and miR-144 and the intensity of luciferase decreased in the presence of miRNAs. Conclusions Based on the results of the present study and since AXL and mTOR genes play a critical role in variety of human cancers, suppression of these genes by their targeting miRNAs, especially miR-34a and miR-144, can be propose as a new strategy for MTC management. However, more studies are needed to approve the hypothesis.

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.

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 Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Wei Na ◽  
Yuxiang Wang ◽  
Pengfei Gong ◽  
Xinyang Zhang ◽  
Ke Zhang ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Reference Genes ◽  
Growth And Development ◽  
Target Genes ◽  
Abdominal Adipose Tissue ◽  
Proliferation And Differentiation ◽  
Quantitative Results ◽  
Tata Box Binding Protein ◽  
Peptidylprolyl Isomerase ◽  
Gene Expression Levels

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

scholarly journals Identification of reliable reference genes for gene expression studies in maternal reproductive tissues and fetal tissues of pregnant cows

2020 ◽  
Author(s):  
Lei Cheng ◽  
Jie Yu ◽  
Xiuzhong Hu ◽  
Min Xiang ◽  
Yu Xia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Related Factor ◽  
Expression Stability ◽  
Relative Expression ◽  
Reproductive Tissues ◽  
Fetal Tissues ◽  
Selection Of

Abstract Background: The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favorable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of ten potential RGs (SUZ12, CNOT11, ACTB, RPL19, RPS9, GAPDH, TBP, HPRT1, SDHA and PPIA) in maternal reproductive tissues and fetal tissues, and to analyze the effect of RG selection on the calculation of the relative expression of target genes. Results: The expression stability of ten potential RGs was analyzed in eight different tissues (caruncular endometrium, intercaruncular endometrium, corpus luteum, ovary, oviduct, mammary gland, embryonic disc and trophoblast) from three pregnant dairy cows. Three programs—GeNorm, NormFinder and Bestkeeper—were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable RGs and least stable RGs. Conclusions: The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

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