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.

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.

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses

Gene ◽  
2015 ◽  
Vol 554 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Vanessa Galli ◽  
Joyce Moura Borowski ◽  
Ellen Cristina Perin ◽  
Rafael da Silva Messias ◽  
Julia Labonde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Real Time Quantitative Pcr ◽  
Accurate Normalization

scholarly journals Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

2011 ◽  
Vol 23 (2) ◽  
pp. 353 ◽  
Author(s):  
Damien B. B. P. Paris ◽  
Ewart W. Kuijk ◽  
Bernard A. J. Roelen ◽  
Tom A. E. Stout
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Embryo Development ◽  
Reference Genes ◽  
Stable Expression ◽  
Pcr Analysis ◽  
Blastocyst Development ◽  
Real Time Quantitative Pcr

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.

scholarly journals Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR

PLoS ONE ◽  
2015 ◽  
Vol 10 (10) ◽  
pp. e0141323 ◽  
Author(s):  
XueYan Li ◽  
JinYun Cheng ◽  
Jing Zhang ◽  
Jaime A. Teixeira da Silva ◽  
ChunXia Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Real Time Quantitative Pcr ◽  
Accurate Normalization ◽  
Lilium Davidii

Identification of Suitable Internal Control Genes for Quantitative Real-Time PCR Expression Analyses in Peanut (Arachis hypogaea)

2010 ◽  
Vol 37 (1) ◽  
pp. 12-19 ◽  
Author(s):  
Yael Brand ◽  
Ran Hovav
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Peanut Kernel ◽  
Tissue Samples ◽  
The Stability

Abstract Real-time qPCR is currently the most sensitive technique available for the detection of low-level mRNA expression. For more reliable and precise gene expression analyses, real-time PCR data for a sequence of interest must be normalized against that of a control gene, which is uniformly expressed in various tissues and during different phases of development. So far, suitable internal controls for gene expression studies in peanut have not been identified. We assessed the expression of 10 frequently used housekeeping genes, specifically ubq10, gapdh, hel1, yls8, 14-3-3, 60s, ubc, ef-1α, act7, and adh3. Using the algorithms available through the GeNorm and NormFinder programs, the stability of their expression was estimated in a set of five diverse peanut tissue samples derived from a Virginia-type peanut cultivar (Shulamit). Collectively, the gene with the most stable expression across all of the examined tissues and both programs was adh3, followed by 60s and yls8, which had minimal estimated intra- and inter-tissue variation. The stability of two stable reference genes (adh3 and yls8) compared with two less stable (14-3-3 and ubq10) reference genes was validated in unpooled tissue samples from five peanut kernel developmental stages. Finally, the effect of the use of one or more reference genes on the observed relative expression levels of an important seed oil metabolism gene, diacylglycerol acyltransferase 1 (Dgat1), during kernel development was demonstrated. Based on findings, the suggestion is that adh3, or a combination of this gene with 60s and yls8 should be considered for use in quantitative mRNA expression analyses in Arachis, particularly in studies involving seed development; whereas ubq10 and gapdh should be avoided.

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