Validation of reference genes for quantitative real-time PCR in chemical exposed and at different age’s brackish water flea 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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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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Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

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.

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Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽

10.7717/peerj.3260 ◽

2017 ◽

Vol 5 ◽

pp. e3260 ◽

Cited By ~ 4

Author(s):

Kai Wang ◽

Yi Niu ◽

Qijun Wang ◽

Haili Liu ◽

Yi Jin ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Reference Genes ◽

Dynamic Range ◽

Expression Profiles ◽

Mrna Levels ◽

Expression Stability ◽

Degenerate Primers ◽

Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

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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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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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Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum

International Journal of Molecular Sciences ◽

10.3390/ijms23020738 ◽

2022 ◽

Vol 23 (2) ◽

pp. 738

Author(s):

Xiu-Mei Dong ◽

Wei Zhang ◽

Shi-Bao Zhang

Keyword(s):

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Molecular Mechanisms ◽

Gene Selection ◽

Expression Profiles ◽

Flower Color ◽

Pcr Analysis ◽

Color Formation ◽

Qrt Pcr

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

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Selection of reference genes for quantitative real-time PCR analysis in halophytic plantRhizophora apiculata

PeerJ ◽

10.7717/peerj.5226 ◽

2018 ◽

Vol 6 ◽

pp. e5226 ◽

Cited By ~ 6

Author(s):

Ankush Ashok Saddhe ◽

Manali Ramakant Malvankar ◽

Kundan Kumar

Keyword(s):

Gene Expression ◽

Salt Stress ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Candidate Reference Gene ◽

Expression Level ◽

Bisphosphate Carboxylase ◽

Qrt Pcr ◽

Selection Of

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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Optimizations for identifying reference genes in bone and cartilage bioengineering

BMC Biotechnology ◽

10.1186/s12896-021-00685-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Fei Xiong ◽

Xiangyun Cheng ◽

Chao Zhang ◽

Roland Manfred Klar ◽

Tao He

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Adipose Tissue ◽

Real Time ◽

Reference Gene ◽

Muscle Tissue ◽

Reference Genes ◽

Target Genes ◽

The Stability ◽

And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

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

Biologia Futura ◽

10.1556/019.70.2019.24 ◽

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.

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