Selection of suitable reference genes for qRT-PCR expression analysis of Codonopsis pilosula under different experimental conditions

2020 ◽  
Vol 47 (6) ◽  
pp. 4169-4181
Author(s):  
Jing Yang ◽  
Xiaozeng Yang ◽  
Zheng Kuang ◽  
Bin Li ◽  
Xiayang Lu ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Reference Genes ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Codonopsis Pilosula ◽  
Suitable Reference ◽  
Selection Of

scholarly journals Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 451 ◽  
Author(s):  
Junchao Zhang ◽  
Wengang Xie ◽  
Xinxuan Yu ◽  
Zongyu Zhang ◽  
Yongqiang Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Expression Stability ◽  
Forage Production ◽  
Experimental Conditions ◽  
Elymus Sibiricus ◽  
Suitable Reference ◽  
Selection Of

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

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.

Selection of suitable reference genes for expression analysis in human glioma using RT-qPCR

2015 ◽  
Vol 123 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Susanne Grube ◽  
Tatjana Göttig ◽  
Diana Freitag ◽  
Christian Ewald ◽  
Rolf Kalff ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Reference Genes ◽  
Human Glioma ◽  
Suitable Reference ◽  
Selection Of

scholarly journals Selection of reference genes for expression analysis of plant-derived microRNAs in Plutella xylostella using qRT-PCR and ddPCR

PLoS ONE ◽  
2019 ◽  
Vol 14 (8) ◽  
pp. e0220475 ◽  
Author(s):  
Lingling Zhang ◽  
Xiaodong Jing ◽  
Wei Chen ◽  
Jianlin Bai ◽  
Liette Vasseur ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Plutella Xylostella ◽  
Reference Genes ◽  
Qrt Pcr ◽  
Selection Of

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.

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