scholarly journals Screening of reference genes in real-time PCR for Radopholus similis

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6253 ◽  
Author(s):  
Jun-Yi Li ◽  
Wan-Zhu Chen ◽  
Si-Hua Yang ◽  
Chun-Ling Xu ◽  
Xin Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Suitable Reference Gene ◽  
Radopholus Similis ◽  
Suitable Reference

Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-β catalytic subunit (β-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.

scholarly journals Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

2018 ◽  
Vol 19 (8) ◽  
pp. 2258 ◽  
Author(s):  
Yuning Hu ◽  
Hongtuo Fu ◽  
Hui Qiao ◽  
Shengming Sun ◽  
Wenyi Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Translation Initiation Factor ◽  
Suitable Reference Gene ◽  
Quantitative Real Time Pcr ◽  
Macrobrachium Nipponense ◽  
The Stability ◽  
Suitable Reference

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

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

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.

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 Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Olawale Samuel Adeyinka ◽  
Bushra Tabassum ◽  
Idrees Ahmad Nasir ◽  
Iqra Yousaf ◽  
Imtiaz Ahmad Sajid ◽  
...  
Keyword(s):  
Reference Gene ◽  
Chilo Partellus ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Stable Reference Gene ◽  
Suitable Reference Gene ◽  
Potential Reference Gene ◽  
Alternate Control ◽  
Suitable Reference

Abstract Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.

scholarly journals Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas oryzae pv. oryzae--infection and melatonin supplementation

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Xian Chen ◽  
Pedro Laborda ◽  
Yan Dong ◽  
Fengquan Liu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Housekeeping Genes ◽  
Suitable Reference Gene ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Rice Plants ◽  
Qrt Pcr ◽  
Suitable Reference

Abstract Exogenous melatonin (MT) was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv. oryzae (Xoo)-caused bacterial blight (BB). However, the accurate comparison of the expression levels across samples was a challenging task. In this work, the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR (qRT-PCR), and algorithms geNorm, NormFinder and BestKeeper. Our results indicated that most reference genes remained stable in Xoo-infected rice plants, while a number of reference genes were affected by MT supplementation. Among all studied genes, the transcript levels of 18S(18S ribosomal RNA) and UBC (Ubiquitin-conjugating enzyme E2) remained unaltered by Xoo infection, while UBC and UBQ5(Ubiquitin 5) were the most stable genes when examining simultaneous Xoo-infection and MT supplementation, demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.

scholarly journals Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

2020 ◽  
Author(s):  
Chaofan Jin ◽  
Weihao Song ◽  
Mengya Wang ◽  
Jie Qi ◽  
Quanqi Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Suitable Reference Gene ◽  
Rt Pcr ◽  
Black Rockfish ◽  
Qrt Pcr ◽  
Sebastes Schlegelii ◽  
Suitable Reference

Abstract Background: The quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used technique that relies on the reference gene for gene expression normalization. Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR. However, most previous studies of fishes adopted reference genes that were commonly used in mammals without validation. Results: In this study, we utilized 89 transcriptome datasets covering early developmental stages and adult tissues, and carried out transcriptome-wide identification and validation of reference genes in Sebastes schlegelii. Finally, 121 candidate reference genes were identified based on four criteria. Eight candidates (METAP2, BTF3L4, EIF5A1, TCTP, UBC, PAIRB, RAB10, and DLD) and four commonly used reference genes (TUBA, ACTB, GAPDH, RPL17) in mammals were selected for validation via qRT- PCR and four statistical methods (delta-Ct, BestKeeper, geNorm, and NormFinder). The results revealed that the candidate reference genes we recommended are more stable than traditionally used ones. Conclusions: This is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish, and lay an important foundation for gene expression analysis in teleost.

scholarly journals Evaluation of Housekeeping Gene Expression Stability in Carnation (Dianthus caryophyllus)

2020 ◽  
Author(s):  
Huolin Luo ◽  
Wenjing Yu ◽  
Yuan Tao ◽  
Jonathan Hrovat ◽  
Ahui Xue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Dianthus Caryophyllus ◽  
Housekeeping Gene ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Suitable Reference Gene ◽  
Expression Stability ◽  
Experimental Conditions

Abstract Background: The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability. A suitable reference gene is an absolute prerequisite for accurate normalization, nevertheless, the frequently-used reference gene was reported unstable under different experimental conditions and causes failure to correctly analyze the expression of the interested gene. Therefore, it is vital to systematically evaluate the expression stability of these candidate reference genes before performing RT-qPCR. Results: In this study, two computational statistical methods were used, including geNorm and NormFinder, in order to determine the expression stability of 12 frequently-used reference genes in Dianthus caryophyllus across different experimental conditions. The results show that the expression stability of candidate genes varies greatly in different sample pools, which again proves the instability of these common housekeeping gene expressions. In general, the expression of UBQ10 (ubiquitin10), EF1a (elongation factor 1A) and TIP41 (TIP41-like family protein) were relatively stable under different experimental conditions, while the expression stability of 18S (18S Ribosome RNA), TIF5A (translation initiation factor 5A) and PP2A (protein phosphatase 2A) were relatively poor. Conclusion: EF1α, TIP41 and UBQ10 were considered the most appropriate reference genes when all samples were put together, while UBQ10 was most stable in exogenous hormone treatments. TUB and UBQ10 can be used as reliable internal control genes under stress, while CYP and TUA can act as reliable internal controls in different tissues. This is the first systematic study of selection of reference genes in Carnation, and will benefit future expression studies in this crop.

scholarly journals Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Madhab Kumar Sen ◽  
Kateřina Hamouzová ◽  
Pavlina Košnarová ◽  
Amit Roy ◽  
Josef Soukup
Keyword(s):  
Reference Gene ◽  
Herbicide Resistance ◽  
Reference Genes ◽  
Gene Selection ◽  
High Yield ◽  
Suitable Reference Gene ◽  
Grass Species ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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