Selection of reference gene in Eucalyptus camaldulensisfor real-time qRT-PCR

Rhizophora apiculatais a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) inR. apiculataphysiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively rankedEF1α followed byACTas the most stable candidate reference genes inR. apiculataphysiological tissues. Moreover, β-TUBand 18S were ranked as moderately stable candidate reference genes, while GAPDH andrbcLwere least stable reference genes. Under salt stress,EF1α was comprehensively recommended top-ranked candidate reference gene followed byACTand 18S. In order to validate the identified most stable candidate reference genes,EF1α,ACT, 18S andUBQwere used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level ofNHXvaried according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR inR. apiculataphysiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes inR. apiculata.

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Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L.

PLoS ONE ◽

10.1371/journal.pone.0226668 ◽

2020 ◽

Vol 15 (2) ◽

pp. e0226668 ◽

Cited By ~ 1

Author(s):

Xu Su ◽

Liuyang Lu ◽

Yashe Li ◽

Congai Zhen ◽

Guilei Hu ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Expression Analysis ◽

Real Time Pcr ◽

Gene Selection ◽

Quantitative Real Time Pcr ◽

Reference Gene Selection ◽

Qrt Pcr ◽

Galium Aparine ◽

Selection For

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

10.21203/rs.3.rs-847536/v1 ◽

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.

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Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Forests ◽

10.3390/f11020193 ◽

2020 ◽

Vol 11 (2) ◽

pp. 193

Author(s):

Dandan Li ◽

Sen Yu ◽

Minzhen Zeng ◽

Xiao Liu ◽

Jia Yang ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Reference Genes ◽

Pcr Analysis ◽

Stable Gene ◽

Larix Olgensis ◽

Qrt Pcr ◽

Study Gene Expression ◽

Selection Of

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

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

Scientific Reports ◽

10.1038/s41598-021-03098-x ◽

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.

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Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽

10.7717/peerj.6536 ◽

2019 ◽

Vol 7 ◽

pp. e6536 ◽

Cited By ~ 5

Author(s):

Li Miao ◽

Xing Qin ◽

Lihong Gao ◽

Qing Li ◽

Shuzhen Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Stable Expression ◽

Quantitative Real Time Pcr ◽

Graft Union ◽

Expression Levels ◽

Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

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Selection of Reference Genes for Quantitative Real-Time PCR in Chrysoperla nipponensis (Neuroptera: Chrysopidae) Under Tissues in Reproduction and Diapause

Journal of Insect Science ◽

10.1093/jisesa/ieaa079 ◽

2020 ◽

Vol 20 (4) ◽

Author(s):

Xiao Wang ◽

Xue Kong ◽

Shaoye Liu ◽

Haiyi Huang ◽

Zhenzhen Chen ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Fat Body ◽

Model Organism ◽

Reproductive Diapause ◽

Genomics Research ◽

Polymerase Chain ◽

Selection Of ◽

Polymerase Chain Reaction Data

Abstract Chrysoperla nipponensis (Okamoto), which has the unique diapause phenotype distinguishable from nondiapause adult, is an ideal model organism for studying the mechanism of reproductive diapause. However, there is no reliable and effective reference genes used for the reproductive diapause study of C. nipponensis. Therefore, in this study, we evaluated the expression stability of 10 candidate reference genes (Tub1, Arpc5, EF1a, 128up, RpS5, RpS26e, GAPDH, Arp3, Actin, α-Tub) in adults under diapause and nondiapause induction conditions using four statistical algorithms including GeNorm, NormFinder, Bestkeeper, and ∆CT method. Results showed that Arp3 and Tub1 were the most stable reference genes in all samples and in the adult tissues group. Arp3 and RpS5 were the most stable reference genes in the development degree group. α-Tub and EF1a were unstable reference genes under the conditions of this study. Meanwhile, to verify the reliability of the reference genes, we evaluated the relative expression levels of Vg and VgR in different treatments. Significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was, respectively, observed when using Arp3 as the reference gene but not when using an unstable reference gene. The reference genes identified in this work provided not only the basis for future functional genomics research in diapause of C. nipponensis and will also identify reliable normalization factors for real-time quantitative real-time polymerase chain reaction data for other related insects.

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Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (<italic>Anser anser domestica</italic>) Gene Expression

Asian-Australasian Journal of Animal Sciences ◽

10.5713/ajas.2012.12417 ◽

2013 ◽

Vol 26 (3) ◽

pp. 423-432 ◽

Cited By ~ 6

Author(s):

Hong Ji ◽

Jianfa Wang ◽

Juxiong Liu ◽

Jingru Guo ◽

Zhongwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Genes ◽

Pcr Analysis ◽

Anser Anser ◽

Qrt Pcr ◽

Selection Of

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

Open Chemistry ◽

10.2478/s11532-012-0164-5 ◽

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

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