Screening of reference genes using real-time quantitative PCR for gene expression studies inNeoseiulus barkeriHughes (Acari: Phytoseiidae)

2018 ◽  
Vol 109 (4) ◽  
pp. 443-452 ◽  
Author(s):  
C. Wang ◽  
J. Yang ◽  
Q. Pan ◽  
S. Yu ◽  
R. Luo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

AbstractA stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms.Neoseiulus barkeriHughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACTandHsp40were the top two stable reference genes in developmental stages. TheHsp60andHsp90were the most stable reference genes in various acaricides stress. For alterations in temperature,Hsp40and α-TUBwere the most suitable reference genes. About UV stress,EF1α and α-TUBwere the best choice, and for the different prey stress, β-ACTand α-TUBwere best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression inN. barkeri.

scholarly journals Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

2011 ◽  
Vol 56 (No. 5) ◽  
pp. 213-216 ◽  
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.

scholarly journals Validation of Reference Genes for Gene Expression Normalization in RAW264.7 Cells under Different Conditions

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Zhenzhen Bao ◽  
Yanli Huang ◽  
Jiyu Chen ◽  
Zhenglong Wang ◽  
Jiang Qian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Raw264.7 Cells ◽  
Antigen Uptake ◽  
Expression Levels ◽  
Real Time Quantitative Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability ◽  
Suitable Reference

RAW264.7 is a macrophage strain derived from mice tumour and shows a significant ability in antigen uptake. Real-time quantitative PCR (RT-qPCR) is one of the most commonly used methods in gene studies and requires suitable reference genes to normalize and quantitate the expression of gene of interest with sensitivity and specificity. However, suitable reference genes in RAW264.7 cells have not yet been identified for accurate gene expression quantification. In the current study, we evaluated expression levels of ten candidate reference genes in RAW264.7 cells under different conditions. RT-qPCR results indicated significant differences in the expression levels among the ten reference genes. Statistical analyses were carried out using geNorm, NormFinder, and BestKeeper software to further investigate the stability of the reference genes. Integrating the results from the three analytical methods, cytochrome c-1 and hydroxymethylbilane synthase were found to be the most stable and therefore more suitable reference genes, while ribosomal protein L4 and cyclophilin A were the least stable. This study emphasises the importance of identifying and selecting the most stable reference genes for normalization and provides a basis for future gene expression studies using RAW264.7 cells.

scholarly journals Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽  
2018 ◽  
Vol 61 (5) ◽  
pp. 349-358 ◽  
Author(s):  
Yanchun You ◽  
Miao Xie ◽  
Liette Vasseur ◽  
Minsheng You
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Plutella Xylostella ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

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 Evaluation of putative reference genes for quantitative real-time PCR normalization inLilium regaleduring development and under stress

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1837 ◽  
Author(s):  
Qiang Liu ◽  
Chi Wei ◽  
Ming-Fang Zhang ◽  
Gui-Xia Jia
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Quantitative Pcr ◽  
Biotic Stress ◽  
Reference Genes ◽  
Developmental Stages ◽  
Real Time Quantitative Pcr ◽  
The Stability ◽  
Experimental Treatments

Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression,Lilium, and particularlyL. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely18S rRNA,ACT,BHLH,CLA,CYP,EF1,GAPDH,SANDandTIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms,BHLHwas superior to the other candidates when all the experimental treatments were analyzed together;CLAandEF1were also recommended by two of the three algorithms. As for specific conditions,EF1under various developmental stages,SANDunder biotic stress,CYP/GAPDHunder drought stress, andTIP41under salinity stress were generally considered suitable. All the algorithms agreed on the stability ofSANDandGAPDHunder cold stress, while onlyCYPwas selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level ofLrLOXin leaves inoculated withB. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding ofLilium.

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.

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