scholarly journals Evaluation of Reference Genes in Glenea cantor (Fabricius) by Using qRT-PCR

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1984
Author(s):  
Ran-Ran Su ◽  
Zhong-Yan Huang ◽  
Chao-Wei Qin ◽  
Xia-Lin Zheng ◽  
Wen Lu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Developmental Stages ◽  
Optimal Number ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Adult Tissues ◽  
Biological Studies ◽  
Main Host ◽  
The Stability ◽  
Suitable Reference

Kapok is the main host of Glenea cantor (Fabricius), which causes serious damage and is difficult to control. In severe cases, it often causes the kapok trees to die continuously, which seriously affects the results of urban landscaping. To provide reference for the functional research on related genes in G. cantor, we screened the stable expression of candidate reference genes at different developmental stages (i.e., eggs, larvae, pupae, and adults), in various adult tissues (i.e., head, thorax, abdomen, feet, antennae, and wings), and sexes (i.e., male pupae, female pupae, male adults, and female adults). In this study, 12 candidate reference genes (i.e., ACTINLIKE, ACTININ, TUB, RPL36, RPL32, RPS20, TBP, GAPDH, 18S rRNA, EF1A1, EF1A2, and UBQ) were evaluated using different adult tissues, developmental stages, and sexes. RefFinder, geNorm, NormFinder, and BestKeeper were used to evaluate and comprehensively analyze the stability of the expression of the candidate reference genes. The results show that RPL32 and EF1A1 were the most suitable reference genes in the different adult tissues, and RPL36 and EF1A1 were best at the different developmental stages. RPL36 and EF1A2 were the best fit for the qRT-PCR reference genes in the different sexes, while RPL36 and EF1A1 were the most appropriate qRT-PCR reference genes in all samples. Results from geNorm showed that the optimal number of reference genes was two. We also surveyed the expression of cellulase at the different developmental stages and in the different adult tissues. Results further verified the reliability of the reference genes, and confirmed the best reference genes under the different experimental conditions. This study provides a useful tool for molecular biological studies on G. cantor.

scholarly journals Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Won-Jae Lee ◽  
Ryoung-Hoon Jeon ◽  
Si-Jung Jang ◽  
Ji-Sung Park ◽  
Seung-Chan Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Optimal Number ◽  
Experimental Conditions ◽  
Pearson’S Correlation ◽  
Qrt Pcr ◽  
Differentiated Cells ◽  
Pearson's Correlation ◽  
Suitable Reference

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson’s correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson’s correlation revealed high correlation (r>0.9) between NF3and NFopt. Overall, the present study showed thatHMBS,YWHAZ,SDHA, andTBPare suitable reference genes for qRT-PCR in porcine MSCs.

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.

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

2016 ◽  
Vol 107 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Y. Tan ◽  
X.-R. Zhou ◽  
B.-P. Pang
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Functional Studies ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

scholarly journals Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1253
Author(s):  
An-Pei Yang ◽  
Yu-Sheng Wang ◽  
Cong Huang ◽  
Zhi-Chuang Lv ◽  
Wan-Xue Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Tuta Absoluta ◽  
Pcr Analysis ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Real Time Quantitative Pcr ◽  
Adult Tissues ◽  
Induced Stresses

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

scholarly journals Selection of suitable reference genes for qRT-PCR studies during SE initial dedifferentiation in cotton of different SE capability

2018 ◽  
Author(s):  
Cao Ai Ping ◽  
Shao Dong Nan ◽  
Cui Bai Ming ◽  
Zheng Yin Ying ◽  
Sun jie
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Expression Level ◽  
Expression Level ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Cotton Species ◽  
Wide Range ◽  
The Stability ◽  
Suitable Reference

Analysis of gene expression level by RNA sequencing (RNA-seq ) has a wide range of biological purposes in various species. Real-time fluorescent quantitative PCR (qRT-PCR) evaluated gene expression levels and validated transcriptomic, which will depend on the stably expressed reference genes for normalization of the gene expression level under specific situations. In this study, 15 candidate genes were selected from transcriptome datasets during somatic embryogenesis (SE) initial dedifferentiation in Gossypium hirsutum L. of different SE capability. To evaluate the stability of those genes, geNorm, NormFinder and BestKeeper were used. The results revealed that ENDO4 and 18srRNA could be as appropriate reference genes under all conditions. The stability and reliability of the reference genes were further tested through comparison of qRT-PCR results and RNA-seq data, as well as evaluation of the expression profiles of auxin-responsive protein (AUX22) and ethylene-responsive transcription factor (ERF17). In summary, the results of our study indicate the most suitable reference genes for qRT-PCR during three induction stages in four cotton species.

scholarly journals Reference Gene Selection for miRNA qRT-PCR Analysis in Lily

2020 ◽  
Author(s):  
Qian Zhang ◽  
Xue Gao ◽  
Lian-Juan Wang ◽  
Yu-Qian Zhao ◽  
Gui-Xia Jia
Keyword(s):  
Reference Gene ◽  
Stress Responses ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Critical Element ◽  
Biotic And Abiotic Stress ◽  
Experimental Conditions ◽  
Qrt Pcr

Abstract Background: The selection of reliable reference genes is a critical element for obtaining accurate gene expression data to assess quantitative real-time polymerase chain reaction (qRT-PCR) performance. It is critical to use suitable reference genes in miRNA qRT-PCR because of short amplification products and large differences in the expression levels of target miRNAs involved in some biological processes. However, in lily, which exhibits a large complex genome but lacks a reference, the available miRNA reference genes for use in qRT-PCR under various treatment conditions are limited, and their reliability has rarely been systematically evaluated.Results: In this study, 8 candidate reference genes, including three classic housekeeping genes and five potential miRNAs from the miRNA library of L. × formolongi, were selected and assessed for expression stability utilizing the BestKeeper, geNorm and Normfinder tools, together with the Delta Ct method, across a diverse set of biotic and abiotic experimental conditions (developmental stages, tissues, heat stress and pathogen defence) to determine the best reference gene(s) for L. × formolongi and L. regale. The final ranking was reordered by using RankAggreg, and the results showed that the novel miRNA PC-3p-67_108977 and the conserved miRNAs miR399a, miR399a and U6 were the most stable genes for L. × formolongi and L. regale, respectively, under all tested experimental conditions. Additionally, PC-3p-67_108977 and U6 were the most suitable genes for qRT-PCR studies in lily.Conclusions: This study provides a comprehensive evaluation of the reliability of reference genes for miRNA studies on development and biotic and abiotic stress responses in different lilies. These results will be beneficial for miRNA identification and functional studies of lilies in the future.

scholarly journals Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Forests ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1217
Author(s):  
Tingting Zhou ◽  
Xiaoming Yang ◽  
Fangfang Fu ◽  
Guibin Wang ◽  
Fuliang Cao
Keyword(s):  
Gene Expression ◽  
Ginkgo Biloba ◽  
Reference Genes ◽  
Developmental Stages ◽  
Deciduous Tree ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Transcriptomic Data ◽  
Suitable Reference ◽  
Selection Of

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

scholarly journals Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression inDioscorea opposita

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Xiting Zhao ◽  
Xiaoli Zhang ◽  
Xiaobo Guo ◽  
Shujie Li ◽  
Linlin Han ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Expression Studies ◽  
Tuber Crop ◽  
Polymerase Chain ◽  
Gene Expression Studies ◽  
Suitable Reference

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea oppositaThunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression ofPE2.1andPE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection inD. oppositaand will contribute toward more accurate gene analysis studies of the genusDioscorea.

scholarly journals Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiaoxin Xie ◽  
Tinghui Liu ◽  
Adel Khashaveh ◽  
Chaoqun Yi ◽  
Xiaoxu Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Target Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Experimental Conditions ◽  
Functional Studies ◽  
Qpcr Analysis ◽  
Hippodamia Variegata ◽  
Convenient Technique ◽  
Suitable Reference

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-β, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

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