Selection and validation of reference genes for quantitative gene expression
            analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

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Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽

10.1139/gen-2017-0176 ◽

2018 ◽

Vol 61 (5) ◽

pp. 349-358 ◽

Cited By ~ 6

Author(s):

Yanchun You ◽

Miao Xie ◽

Liette Vasseur ◽

Minsheng You

Keyword(s):

Gene Expression ◽

Real Time ◽

Plutella Xylostella ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

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Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

Biology Methods and Protocols ◽

10.1093/biomethods/bpw005 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Roshini Kalagara ◽

Weimin Gao ◽

Honor L. Glenn ◽

Colleen Ziegler ◽

Laura Belmont ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Stable Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using Ct value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.

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Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

Bulletin of Entomological Research ◽

10.1017/s000748531800072x ◽

2018 ◽

Vol 109 (4) ◽

pp. 443-452 ◽

Cited By ~ 1

Author(s):

C. Wang ◽

J. Yang ◽

Q. Pan ◽

S. Yu ◽

R. Luo ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Genes ◽

Developmental Stages ◽

Quantitative Polymerase Chain Reaction ◽

Real Time Quantitative Pcr ◽

Expression Studies ◽

Gene Expression Studies ◽

The Stability ◽

Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

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Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies

Clinical Chemistry ◽

10.1373/clinchem.2013.209478 ◽

2014 ◽

Vol 60 (3) ◽

pp. 451-454 ◽

Cited By ~ 11

Author(s):

Liesbeth Ceelen ◽

Jurgen De Craene ◽

Ward De Spiegelaere

Keyword(s):

Gene Expression ◽

Cell Line ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Heparg Cell ◽

Expression Studies ◽

Heparg Cell Line ◽

Heparg Cells ◽

Gene Expression Studies

Abstract BACKGROUND The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. SUMMARY In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

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Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

PLoS ONE ◽

10.1371/journal.pone.0246227 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0246227

Author(s):

Nicole Dittrich Hosni ◽

Ana Carolina Anauate ◽

Mirian Aparecida Boim

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

High Glucose ◽

Real Time Pcr ◽

Reference Genes ◽

Mesangial Cell ◽

Mesangial Cells ◽

Expression Studies ◽

Gene Expression Studies

Background Real-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high-quality qPCR, providing strong support for results, and performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used thus far to enhance the validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g., losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cell and podocyte studies of an in vitro DKD model of disease and its treatment. Methods Five software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted, and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n = 5; 5 mM glucose), mannitol (n = 5; 30 mM, as osmotic control), HG (n = 5; 30 mM glucose), and HG + losartan (n = 5; 30 mM glucose and 10−4 mM losartan). Real-time PCR was performed according to MIQE guidelines. Results We identified that the use of 2 genes was the best combination for qPCR normalization for both mesangial cells and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results. Conclusion This analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR, while for podocytes, HPRT and GAPDH should be chosen.

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Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

Czech Journal of Animal Science ◽

10.17221/1428-cjas ◽

2011 ◽

Vol 56 (No. 5) ◽

pp. 213-216 ◽

Cited By ~ 2

Author(s):

M. Nesvadbová ◽

A. Knoll

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Muscle Tissue ◽

Reference Genes ◽

Expression Profiles ◽

Quantitative Polymerase Chain Reaction ◽

Expression Studies ◽

Gene Expression Studies ◽

The Stability

The selection of reference genes is essential for gene expression studies when using a real-time quantitative polymerase chain reaction (PCR). Reference gene selection should be performed for each experiment because the gene expression level may be changed in different experimental conditions. In this study, the stability of mRNA expression was determined for seven genes: HPRT1, RPS18, NACA, TBP, TAF4B, RPL32 and OAZ1. The stability of these reference genes was investigated in the skeletal muscle tissue of pig foetuses, piglets and adult pigs using real-time quantitative PCR and SYBR green chemistry. The expression of stability of the used reference genes was calculated using the geNorm application. Different gene expression profiles among the age categories of pigs were found out. RPS18 has been identified as the gene with the most stable expression in the muscle tissue of all pig age categories. HPRT1 and RPL32 were found to have the highest stability in piglets and adult pigs, and in foetuses and adults pigs, respectively. The newly used reference gene, TAF4B, reached the highest expression stability in piglets.

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Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

10.1101/2021.01.19.427251 ◽

2021 ◽

Author(s):

Nicole Dittrich Hosni ◽

Ana Carolina Anauate ◽

Mirian Aparecida Boim

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

High Glucose ◽

Real Time Pcr ◽

Reference Genes ◽

Mesangial Cell ◽

Mesangial Cells ◽

Expression Studies ◽

Gene Expression Studies

ABSTRACTBackgroundReal-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high quality qPCR, providing strong support for results, as well as performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used so far to enhance validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g. Losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cells and podocytes studies of an in vitro DKD model of disease and its treatment.MethodsFive software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n=5; 5mM glucose), mannitol (n=5; 30mM, as osmotic control), HG (n=5; 30mM glucose), and HG + losartan (n=5; 30mM glucose and 10-4 mM of losartan). Real-time PCR was performed according to MIQE guidelines.ResultsWe identified that the use of 2 genes is the best combination for qPCR normalization for both mesangial cell and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results.ConclusionThis analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR technique, while for podocytes, HPRT and GAPDH should be chosen.

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Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PLoS ONE ◽

10.1371/journal.pone.0046451 ◽

2012 ◽

Vol 7 (9) ◽

pp. e46451 ◽

Cited By ~ 176

Author(s):

Deshui Liu ◽

Lindan Shi ◽

Chenggui Han ◽

Jialin Yu ◽

Dawei Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Nicotiana Benthamiana ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Expression Studies ◽

Gene Expression Studies

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Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

Scientific Reports ◽

10.1038/s41598-021-88151-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Meng Wang ◽

Tingting Ren ◽

Prince Marowa ◽

Haina Du ◽

Zongchang Xu

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Suitable Reference Gene ◽

Normalize Gene Expression ◽

Suaeda Glauca ◽

Suitable Reference ◽

Germinating Seeds ◽

Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

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Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

10.21203/rs.3.rs-145449/v1 ◽

2021 ◽

Author(s):

Zhongyi Yang ◽

Rui Zhang ◽

Zhichun Zhou

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Geometric Mean ◽

Gene Transcript ◽

Expression Stability ◽

Schima Superba ◽

Expression Studies ◽

Gene Expression Studies ◽

Different Tissues

Abstract Background Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

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