scholarly journals Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Xu Zhao ◽  
Huanling Yang ◽  
Mingjie Chen ◽  
Xiaoxia Song ◽  
Changxia Yu ◽  
...  
Keyword(s):  
High Temperature ◽  
Real Time ◽  
Temperature Stress ◽  
Reference Gene ◽  
Reference Genes ◽  
Candidate Reference Gene ◽  
Housekeeping Genes ◽  
High Temperature Stress ◽  
Stable Gene ◽  
Mrna Levels

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes,TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC,andH2A, were tested for their adequacy inLentinula edodes(L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia ofL. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm,TUBhad the lowest M values inL. edodesstrains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 wasTUB(0.030), and the best candidate reference gene in strain 18N44 wasUBI(0.047). In BestKeeper analysis, the standard deviation (SD) values ofUBC,TUA,H2A,EF1,ACT,18S, andGTPin strain 18 and those ofGADPHandGTPin strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, onlyUBIandTUBwere found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses,TUBwas the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction inL. edodesmycelia under high-temperature stress.

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

2019 ◽  
Vol 70 (4) ◽  
pp. 261-267
Author(s):  
Gaigai Du ◽  
Liyuan Wang ◽  
Huawei Li ◽  
Peng Sun ◽  
Jianmin Fu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Suitable Reference Gene ◽  
Diospyros Kaki ◽  
Stable Gene ◽  
Expression Studies ◽  
Gene Expression Studies

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.

scholarly journals Validation of Reference Genes for Quantitative Real-time PCR in Diaphanosoma Celebensis: For Chemical Exposure Conditions and Different Ages

2021 ◽  
Author(s):  
Young-Mi Lee ◽  
Soyeon In ◽  
Se-Joo Kim ◽  
Eun-Ji Won ◽  
Hayoung Cho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Expression Profiles ◽  
Chemical Exposure ◽  
Mrna Levels ◽  
Qrt Pcr ◽  
Different Ages ◽  
Diaphanosoma Celebensis

Abstract Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to rule out variations in gene expression among samples. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (e.g., adult), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

scholarly journals Validation of reference genes for quantitative real-time PCR in chemical exposed and at different age’s brackish water flea Diaphanosoma celebensis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Young-Mi Lee ◽  
Hayoung Cho ◽  
Ryeo-Ok Kim ◽  
Soyeon In ◽  
Se-Joo Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Brackish Water ◽  
Reference Genes ◽  
Expression Profiles ◽  
Mrna Levels ◽  
Water Flea ◽  
Qrt Pcr ◽  
Diaphanosoma Celebensis

AbstractReal-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to confirm relative gene expression levels by comparison, and rule out variations that might occur in analytical procedures. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (H2A was stable, whereas Act was fairly unstable in adults), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

Selection of reference genes for quantitative real-time RT-PCR on gene expression in Golden Pompano (Trachinotus ovatus)

2017 ◽  
Vol 20 (3) ◽  
pp. 583-594 ◽  
Author(s):  
X.J. Chen ◽  
X.Q. Zhang ◽  
S. Huang ◽  
Z.J. Cao ◽  
Q.W. Qin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Housekeeping Genes ◽  
Poly I:C ◽  
Stable Gene ◽  
Trachinotus Ovatus ◽  
Golden Pompano ◽  
Normal Physiological Condition ◽  
Selection Of

Abstract Golden pompano (Trachinotus ovatus) is an important economically fish species. In this study, with an aim to identify reliable reference genes for quantitative real-time PCR (qRT-PCR) in golden pompano, we evaluated the expression stability of eight housekeeping genes in the presence and absence of poly I:C stimulation in eight tissues. The PCR data was analyzed by geNorm and NormFinder algorithms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations. When under normal physiological condition, geNorm and NormFinder identified B2M and 18S as suitable genes. When studying gene expression under conditions of poly I:C stimulation, the selection of the internal controls should be selected on a tissue basis. At 12 h stimulation, geNorm ranked Actin/UBCE, Actin/B2M, UBCE/B2M, Actin/UBCE, RPL13/B2M, UBCE/GAPDH, B2M/RPL13, and UBCE/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, intestine, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 48 h stimulation. Taken together, these results indicate that B2M and 18S are the most stable gene across tissue types under normal physiological conditions. However, during poly I:C stimulation, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types. If one gene is preferred, B2M, B2M, UBCE, Actin, B2M/RPL13, B2M, B2M, and RPL13 may be used in spleen, kidney, liver, gill, intestine, brain, muscle, and heart of golden pompano, respectively.

scholarly journals Reference Gene Validation for Quantitative Real-time PCR Studies in Amphibian Kidney-derived A6 Epithelial Cells

2019 ◽  
Vol 47 (2) ◽  
pp. 63-70 ◽  
Author(s):  
Elin Verbrugghe ◽  
An Martel ◽  
Frank Pasmans
Keyword(s):  
Cell Line ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Tissue Sample ◽  
Housekeeping Genes ◽  
Optimal Number ◽  
Pairwise Variation ◽  
Expression Stability ◽  
Specific Tissue

Quantitative real-time polymerase chain reaction is a widely used technique that relies on reference genes for the normalisation of gene expression. These reference genes are constitutively expressed and must remain stable across all samples and treatments. Stability of housekeeping genes may vary and must be optimised for a specific tissue, sample or cell line. Here we present a study screening for possible reference gene candidates, eef1a1, rpl8, sub1.L, clta, H4 and odc1, in the Xenopus laevis (A6) kidney cell line. Quantification cycle results were analysed using geNorm to calculate the average expression stability and the coefficient of variation (CV) for each candidate reference gene. All of the tested genes met the guidelines for stable reference genes, namely an average expression stability of < 0.5 and a CV value of < 0.2, with eef1a1 > sub1.L > rpl8 > clta > odc1 > H4. By using pairwise variation analysis, the optimal number of reference targets was determined to be 2. As such, we report that the reference genes eef1a1 and sub1.L should be used to achieve optimal normalisation in A6 cells.

scholarly journals Selection of reference genes for quantitative real-time PCR evaluation of chronic erythropoietin treatment effect on the SH-SY5Y and PC12 cells

2013 ◽  
Vol 11 (3) ◽  
pp. 348-357
Author(s):  
Klemen Španinger ◽  
Arthur Sytkowski ◽  
Nataša Debeljak
Keyword(s):  
Real Time ◽  
Pc12 Cells ◽  
Reference Gene ◽  
Reference Genes ◽  
Housekeeping Genes ◽  
Expression Stability ◽  
Normalization Gene ◽  
Erythropoietin Treatment ◽  
Control Plate ◽  
Selection Of

AbstractAbstract The quantitative real-time polymerase chain reaction (qPCR) is a sensitive technique for examining the influence of erythropoietin (Epo) on gene expression. A critical and fundamental step for data analysis is the selection of and normalization to the optimal reference gene(s). We identified appropriate reference gene(s) among 32 genes during chronic recombinant human Epo (rHuEpo) treatment of SH-SY5Y cells using TaqMan human Express Endogenous Control Plate. Expression stability of the selected reference gene (RPLP) was retested with qPCR, together with two commonly used reference genes (GAPDH, ACTB) and six genes of interest (EPOR, EPO, STAT5B, STAT5A, JUN, AKT). In PC12 cells, three commonly used reference genes (Gapdh, CycA and Ywhaz) and seven genes of interest (EpoR, Epo, Stat5b, Stat5a, Jun, Akt, Fos) were evaluated. For the evaluation of expression stability, geNorm, NormFinder and BestKeeper software were used. All three gave similar results. We demonstrated that among the housekeeping genes, RPLP in SH-SY5Y and CycA and Ywhaz in PC12 are the most stable genes. Additionally, we showed that normalization with GAPDH gave misleading results compared to normalization with geNorm. In conclusion, selection of the appropriate normalization gene(s) is crucial for correct interpretation of rHuEpo treatment results. Graphical abstract

scholarly journals Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142015 ◽  
Author(s):  
Heng Xu ◽  
Jian-Dong Bao ◽  
Ji-Song Dai ◽  
Yongqing Li ◽  
Ying Zhu
Keyword(s):  
High Temperature ◽  
Temperature Stress ◽  
Reference Genes ◽  
High Temperature Stress ◽  
Rice Endosperm ◽  
Qrt Pcr ◽  
Genome Wide
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