scholarly journals Selection of suitable reference genes for qRT-PCR normalisation under different experimental conditions in Eucommia ulmoides Oliv

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Jing Ye ◽  
Cang-Fu Jin ◽  
Nan Li ◽  
Min-Hao Liu ◽  
Zhao-Xue Fei ◽  
...  
Keyword(s):  
Reference Genes ◽  
Eucommia Ulmoides ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Suitable Reference ◽  
Selection Of

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.

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.

Selection and validation of reference genes for quantitative real-time PCR analysis of Nitraria tangutorum

2020 ◽  
Author(s):  
Bo Wang ◽  
Lirong WANG ◽  
Huirong Duan ◽  
Peifang Chong ◽  
Shiping Su ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Expression Stability ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Nitraria Tangutorum ◽  
Desert Areas ◽  
Polymerase Chain ◽  
Suitable Reference

Abstract Background: Suitable reference genes can be used to calibrate the error in quantitative real‑time polymerase chain reaction (qRT-PCR) experiments and make the results more credible. However, reference genes suitable for different species and different experimental conditions do not exist. Nitraria tangutorum Bobr. is a typical plant in desert areas and desert plains, which is drought-resistant, saline-alkali resistant, barren-resistant, and has extremely strong adaptability. Due to insufficient understanding of the importance of this germplasm in the past, it is still unclear which genes can be used as reference genes to calibrate qRT-PCR data of N. tangutorum .Results: In this study, we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in three tissues (root, stem and leaf) and under five abiotic stresses (salt, drought, heat, cold, and ABA) of N. tangutorum seedlings by qRT-PCR. Three analysis software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate expression stability of ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of the waxy synthesis of N. tangutorum, verified the accuracy of the experimental results.Conclusion: Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This study provides the first data on stable reference genes in N. tangutorum, which will be beneficial to study of the gene expression of N. tangutorum and other Nitraria species in the future.

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 and validation of reference genes for quantitative gene expression normalization in Taxus spp.

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaikai Zhang ◽  
Wei Fan ◽  
Duanfen Chen ◽  
Luyuan Jiang ◽  
Yunfeng Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Reference Genes ◽  
Expression Patterns ◽  
Future Research ◽  
Taxus Chinensis ◽  
Experimental Conditions ◽  
Taxol Biosynthesis ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractQuantitative real-time PCR (qRT-PCR) is commonly used to measure gene expression to further explore gene function, while suitable reference genes must be stably expressed under different experimental conditions to obtain accurate and reproducible data for relative quantification. Taxol or paclitaxel is an important anticancer compound mainly identified in Taxus spp. The molecular mechanism of the regulation of taxol biosynthesis is current research goal. However, in the case of Taxus spp., few reports were published on screening suitable reference genes as internal controls for qRT-PCR. Here, eight reference genes were selected as candidate reference genes for further study. Common statistical algorithms geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to analyze the data from samples collected from a cell line of Taxus × media under various experimental conditions and from tissues of Taxus chinensis var. mairei. The expression patterns of TcMYC under salicylic acid treatment differed significantly, with the best and worst reference genes in the cell line. This study screened out suitable reference genes (GAPDH1 and SAND) under different treatments and tissues for the accurate and reliable normalization of the qRT-PCR expression data of Taxus spp. At the same time, this study will aid future research on taxol biosynthesis-related genes expression in Taxus spp., and can also be directly used to other related species.

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.

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