scholarly journals Reference genes selection for transcript normalization in kenaf (Hibiscus cannabinusL.) under salinity and drought stress

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1347 ◽  
Author(s):  
Xiaoping Niu ◽  
Jianmin Qi ◽  
Meixia Chen ◽  
Gaoyang Zhang ◽  
Aifen Tao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
18S Rrna ◽  
Hibiscus Cannabinus ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Yield And Quality ◽  
Selection For ◽  
Transcriptomic Level

Kenaf (Hibiscus cannabinus) is an economic and ecological fiber crop but suffers severe losses in fiber yield and quality under the stressful conditions of excess salinity and drought. To explore the mechanisms by which kenaf responds to excess salinity and drought, gene expression was performed at the transcriptomic level using RNA-seq. Thus, it is crucial to have a suitable set of reference genes to normalize target gene expression in kenaf under different conditions using real-time quantitative reverse transcription-PCR (qRT-PCR). In this study, we selected 10 candidate reference genes from the kenaf transcriptome and assessed their expression stabilities by qRT-PCR in 14 NaCl- and PEG-treated samples using geNorm, NormFinder, and BestKeeper. The results indicated thatTUBαand 18S rRNA were the optimum reference genes under conditions of excess salinity and drought in kenaf. Moreover,TUBαand 18S rRNA were used singly or in combination as reference genes to validate the expression levels of WRKY28 and WRKY32 in NaCl- and PEG-treated samples by qRT-PCR. The results further proved the reliability of the two selected reference genes. This work will benefit future studies on gene expression and lead to a better understanding of responses to excess salinity and drought in kenaf.

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 Selecting optimal reference genes for breast cancer research

2020 ◽  
Author(s):  
Qiang Song ◽  
Man Huang ◽  
Guicheng Wu ◽  
Lu Dou ◽  
Wenjin Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Research ◽  
Cell Lines ◽  
Breast Tumor ◽  
Reference Genes ◽  
Breast Cancer Research ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Cancer Tissues

Abstract Background Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes (RGs) is critical for normalizing and evaluating changes in the expression of target genes. However, uniform and reliable RGs for breast cancer research have not been identified, limiting the value of target gene expression studies. Here, we provide a novel approach for mining RGs by using the RNA-seq dataset to identify reliable and accurate RGs that can be applied to different types of breast cancer tissues and cell lines. Methods First, we compiled the transcriptome profiling data from the TCGA database involving 1217 samples to identify novel RGs and then ten genes (SF1, TARDBP, THRAP3, QRICH1, TRA2B, SRSF3, YY1, DNAJC8, RNF10, and RHOA) with relatively stable expression levels were chosen as novel candidate RGs. Additionally, six conventional RGs (ACTB, TUBA1A, RPL13A, B2M, GAPDH, and GUSB) were also selected. To determine and validate the optimal RGs we performed qRT-PCR experiments on 87 samples from 5 types of surgically excised breast tumor specimens including HR+HER2-, HR+HER2+, HR-HER2-, HR-HER2+, breast cancer after neoadjuvant chemotherapy (NAC) and their matched para-carcinoma tissues, furthermore, we also included a benign breast tumor sample. Six biological replicates were included for each tissue. Moreover, we assessed 7 breast cancer cell lines (MCF-10A, MCF-7, T-47D, MDA-MB-231, MDA-MB-468, as well as MDA-MB-231 with either CNR2 knockdown or overexpression; 3 biological replicates for each line). Five statistical algorithms (geNorm, NormFinder, ΔCt method, BestKeeper, and ComprFinder) were used to assess the stability of expression of each RG across all breast cancer tissues and cell lines. Results Our results show that RG combinations SF1+TRA2B+THRAP3 and THRAP3+RHOA+QRICH1 showed stable expression in breast cancer tissues and cell lines, respectively, and that these two combinations displayed good interchangeability. Therefore, we propose that the above two combinations are optimal triplet RGs for breast cancer research. Conclusions In summary, we identified novel and reliable RG combinations for breast cancer research based on a public RNA-seq dataset which lays a solid foundation for accurate normalization of qRT-PCR results across different breast cancer tissues and cells.

scholarly journals Selection of suitable reference genes for qRT-PCR studies during SE initial dedifferentiation in cotton of different SE capability

2018 ◽  
Author(s):  
Cao Ai Ping ◽  
Shao Dong Nan ◽  
Cui Bai Ming ◽  
Zheng Yin Ying ◽  
Sun jie
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Expression Level ◽  
Expression Level ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Cotton Species ◽  
Wide Range ◽  
The Stability ◽  
Suitable Reference

Analysis of gene expression level by RNA sequencing (RNA-seq ) has a wide range of biological purposes in various species. Real-time fluorescent quantitative PCR (qRT-PCR) evaluated gene expression levels and validated transcriptomic, which will depend on the stably expressed reference genes for normalization of the gene expression level under specific situations. In this study, 15 candidate genes were selected from transcriptome datasets during somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum L. of different SE capability. To evaluate the stability of those genes, geNorm, NormFinder and BestKeeper were used. The results revealed that ENDO4 and 18srRNA could be as appropriate reference genes under all conditions. The stability and reliability of the reference genes were further tested through comparison of qRT-PCR results and RNA-seq data, as well as evaluation of the expression profiles of auxin-responsive protein (AUX22) and ethylene-responsive transcription factor (ERF17). In summary, the results of our study indicate the most suitable reference genes for qRT-PCR during three induction stages in four cotton species.

Identification of suitable reference genes of Scylla paramamosain for gene expression profiling in various tissues and under vibrio challenge

Crustaceana ◽  
2018 ◽  
Vol 91 (10) ◽  
pp. 1195-1210 ◽  
Author(s):  
Yabo Fang ◽  
Le Diao ◽  
Fengying Zhang ◽  
Lingbo Ma ◽  
Mengdi Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
18S Rrna ◽  
Elongation Factor ◽  
Scylla Paramamosain ◽  
Mud Crab ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Elongation Factor 1

Abstract The quantitative real-time transcription-polymerase chain reaction (qRT-PCR) is now used widely in studies about mRNA expression levels. The selection of one or more stable reference gene(s) used for data normalization is substantial. In this study, expression levels of eleven candidate reference genes (β-actin, 16S rRNA, 18S rRNA, 28S rRNA, α-I tubulin, GAPDH, ribosomal protein L13, elongation factor 1 α, elongation factor 2, arginine kinase and ubiquitin) were examined using the GenomeLab GeXP analysis system (Beckman Coulter). Gene expression data were analysed using two different statistical models: geNorm and NormFinder. (1) In six different tissues (hepatopancreas, haemocytes, heart, gill, muscle, and testis) from the mud crab, Scylla paramamosain, 18S rRNA and elongation factor 1 α were identified as the two best reference genes. (2) In the haemocytes after being challenged by Vibro parahaemolyticus, the result suggested that ubiquitin was the most stable gene after the treatment. 18S rRNA, elongation factor 1 α and ubiquitin are herein recommended as the best combination. These results provide useful options for reference gene selection under different experimental conditions in qRT-PCR studies in the mud crab.

scholarly journals Screening the Reference Genes for Quantitative Gene Expression by RT-qPCR During SE Initial Dedifferentiation in Four Gossypium hirsutum Cultivars that Have Different SE Capability

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 497 ◽  
Author(s):  
Aiping Cao ◽  
Dongnan Shao ◽  
Baiming Cui ◽  
Xuecheng Tong ◽  
Yinying Zheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gossypium Hirsutum ◽  
Internal Control ◽  
Reference Genes ◽  
18S Rrna ◽  
Strong Positive Correlation ◽  
Rna Seq ◽  
Wide Range ◽  
Qpcr Normalization ◽  
Dedifferentiation Process

RNA sequencing (RNA-Seq)-based gene expression analysis is applicable to a wide range of biological purposes in various species. Reverse transcription quantitative PCR (RT-qPCR) is also used to assess target gene expression utilizing stably expressed reference genes as internal control under a given set of conditions. However, investigations of the reference genes for RT-qPCR normalization in the process of somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum are rarely reported. In this study, on the basis of our previous transcriptome data of three different induction stages during SE initial dedifferentiation process in four G. hirsutum cultivars that have different SE capability, 15 candidate genes were selected during SE initial dedifferentiation process, and their expression stability was evaluated by geNorm, NormFinder, and BestKeeper. The results indicated that the two genes of endonuclease 4 (ENDO4) and 18S ribosomal RNA (18S rRNA) showed stable expression in the four different G. hirsutum cultivars, endowing them to be appropriate reference genes during three induction stages in the four cotton cultivars. In addition, the stability and reliability of the two reference genes of ENDO4 and 18S rRNA were further verified by comparing the expressions of auxin-responsive protein 22 (AUX22) and ethylene-responsive transcription factor 17 (ERF17) between RT-qPCR results and the RNA-seq data, which showed strong positive correlation coefficient (R2 = 0.8396–0.9984), validating again the steady expression of ENDO4 and 18S rRNA as the reliable reference genes. Our results provide effective reference genes for RT-qPCR normalization during SE process in different G. hirsutum cultivars.

Identification of reference genes for qRT-PCR studies of gene expression in giant cells and syncytia induced in Arabidopsis thaliana by Meloidogyne incognita and Heterodera schachtii

Nematology ◽  
2007 ◽  
Vol 9 (3) ◽  
pp. 317-323 ◽  
Author(s):  
Florian Grundler ◽  
Julia Hofmann
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Reference Genes ◽  
18S Rrna ◽  
Housekeeping Genes ◽  
Nematode Infection ◽  
Heterodera Schachtii ◽  
Parasitic Nematodes ◽  
Root Knot Nematode ◽  
Qrt Pcr

AbstractSedentary plant-parasitic nematodes, such as cyst and root-knot nematodes, induce feeding structures in the host root that undergo extensive changes in the gene expression. This phenomenon has previously been studied by gene chip analysis and qRT-PCR. Housekeeping genes are often used routinely as internal references for relative qRT-PCR analyses. However, due to the strong influence of nematode infection on host cell metabolism and physiology, expression of housekeeping genes may be altered considerably, thus limiting reliability of qRT-PCR analyses. Therefore, in the present work we tested UBQ10, ACT2, EF1a, UBP22 and 18S rRNA as potential candidates for relative qRT-PCR studies of gene expression in nematode infection sites in roots of Arabidopsis thaliana. Among the tested candidates only UBP22 and 18S rRNA were stably expressed and, therefore, are reliable reference genes for studying cyst and root-knot nematode infections.

scholarly journals Selection of suitable reference genes for qRT-PCR studies during SE initial dedifferentiation in cotton of different SE capability

2018 ◽  
Author(s):  
Cao Ai Ping ◽  
Shao Dong Nan ◽  
Cui Bai Ming ◽  
Zheng Yin Ying ◽  
Sun jie
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Expression Level ◽  
Expression Level ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Cotton Species ◽  
Wide Range ◽  
The Stability ◽  
Suitable Reference

Analysis of gene expression level by RNA sequencing (RNA-seq ) has a wide range of biological purposes in various species. Real-time fluorescent quantitative PCR (qRT-PCR) evaluated gene expression levels and validated transcriptomic, which will depend on the stably expressed reference genes for normalization of the gene expression level under specific situations. In this study, 15 candidate genes were selected from transcriptome datasets during somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum L. of different SE capability. To evaluate the stability of those genes, geNorm, NormFinder and BestKeeper were used. The results revealed that ENDO4 and 18srRNA could be as appropriate reference genes under all conditions. The stability and reliability of the reference genes were further tested through comparison of qRT-PCR results and RNA-seq data, as well as evaluation of the expression profiles of auxin-responsive protein (AUX22) and ethylene-responsive transcription factor (ERF17). In summary, the results of our study indicate the most suitable reference genes for qRT-PCR during three induction stages in four cotton species.

scholarly journals Public transcriptome database-based selection and validation of reliable reference genes for breast cancer research

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Qiang Song ◽  
Lu Dou ◽  
Wenjin Zhang ◽  
Yang Peng ◽  
Man Huang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Research ◽  
Cell Lines ◽  
Reference Genes ◽  
Breast Cancer Research ◽  
Stable Expression ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Cancer Tissues

Abstract Background Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes (RGs) is critical for normalizing and evaluating changes in the expression of target genes. However, uniform and reliable RGs for breast cancer research have not been identified, limiting the value of target gene expression studies. Here, we aimed to identify reliable and accurate RGs for breast cancer tissues and cell lines using the RNA-seq dataset. Methods First, we compiled the transcriptome profiling data from the TCGA database involving 1217 samples to identify novel RGs. Next, ten genes with relatively stable expression levels were chosen as novel candidate RGs, together with six conventional RGs. To determine and validate the optimal RGs we performed qRT-PCR experiments on 87 samples from 11 types of surgically excised breast tumor specimens (n = 66) and seven breast cancer cell lines (n = 21). Five publicly available algorithms (geNorm, NormFinder, ΔCt method, BestKeeper, and ComprFinder) were used to assess the expression stability of each RG across all breast cancer tissues and cell lines. Results Our results show that RG combinations SF1 + TRA2B + THRAP3 and THRAP3 + RHOA + QRICH1 showed stable expression in breast cancer tissues and cell lines, respectively, and that they displayed good interchangeability. We propose that these combinations are optimal triplet RGs for breast cancer research. Conclusions In summary, we identified novel and reliable RG combinations for breast cancer research based on a public RNA-seq dataset. Our results lay a solid foundation for the accurate normalization of qRT-PCR results across different breast cancer tissues and cells.

scholarly journals Reference Gene Selection for Expression Analyses by qRT-PCR in Dendroctonus valens

Insects ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 328 ◽  
Author(s):  
Chunchun Zheng ◽  
Dongfang Zhao ◽  
Yabei Xu ◽  
Fengming Shi ◽  
Shixiang Zong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
18S Rrna ◽  
Gene Selection ◽  
Expression Levels ◽  
Real Time Quantitative Pcr ◽  
Dendroctonus Valens ◽  
Temperature Conditions ◽  
Selection For ◽  
Suitable Reference

Dendroctonus valens is the main pest of the genus Pinus. To facilitate gene expression analyses, suitable reference genes for adults and mature larvae of D. valens under different temperature conditions were determined. In particular, we obtained the sequences of candidate reference genes, ACT, TUB, SHDA, PRS18, 18S rRNA, and CYP4G55, from transcriptome data. Real-time quantitative PCR was used to analyze gene expression, and geNorm, NormFinder, and BestKeeper were used to evaluate expression stability. Under different temperature conditions, the expression levels of 18S rRNA, PRS18, and TUB were stable in adults, in which 18S rRNA > PRS18 > TUB. In mature larvae, the expression levels of TUB, 18S rRNA, and SDHA were stable, in which TUB > 18S rRNA > SDHA. The combination of 18S rRNA and PRS18 is recommended for studies of gene expression in adults and the combination of 18S rRNA and TUB is effective for studies of gene expression in mature larvae of D. valens under different temperature conditions.

scholarly journals Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tingting Li ◽  
Weigao Yuan ◽  
Shuai Qiu ◽  
Jisen Shi
Keyword(s):  
Gene Expression ◽  
Somatic Embryogenesis ◽  
Reference Genes ◽  
Gene Selection ◽  
Elongation Factor ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Suitable Reference ◽  
Functional Analyses ◽  
Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

Sign in / Sign up

Export Citation Format

Share Document