Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization.

IF 1.9 3区 生物学 Q2 BIOLOGY
Journal of Biological Research-Thessaloniki Pub Date : 2016-07-19 eCollection Date: 2016-12-01 DOI:10.1186/s40709-016-0053-8
Seyyed Hamidreza Hashemi, Ghorbanali Nematzadeh, Gholamreza Ahmadian, Ahad Yamchi, Markus Kuhlmann
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引用次数: 15

Abstract

Background: The use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis-halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted.

Results: Ten candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae.

Conclusions: Overall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A. littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.

Abstract Image

Abstract Image

Abstract Image

实时荧光定量PCR归一化的海雀内参基因鉴定与验证。
背景:使用稳定表达的基因作为正常化因子在定量实时聚合酶链反应(qPCR)准确可靠的表达分析中起着至关重要的作用。最近的研究表明,在不同的组织和实验条件下,常见管家基因的表达水平是不同的。RNA样品中的基因组DNA污染也是影响qPCR数据解释的另一个重要因素。据估计,基因表达分析中的dna污染导致了对RNA转录水平的高估。本研究的目的是验证盐胁迫和恢复条件下滨海海茅两种不同组织中最稳定表达的内参基因。此外,还进行了基于qpcr的dna污染监测方法。结果:在根和叶组织、盐胁迫和恢复条件4组样品中,分析了10个参与不同生物过程的候选内参基因。为了确定最稳定表达的内参基因,采用了三种统计方法(geNorm、NormFinder和BestKeeper)。根据得到的结果,根据10个候选内参基因的表达稳定性进行排序。本研究结果表明,RPS3、EF1A、GTF和RPS12等4个内参基因可作为所有选择条件和组织的一般内参基因。同时,针对不同的组织和条件提出了4组内参基因,分别为:盐胁迫和根系样品的RPS3、EF1A和UBQ;RPS3、EF1A、UBQ、GAPDH为恢复状态;叶片样品的U2SURP和GTF。此外,为了评估RNA样品中的DNA污染,基于核糖体DNA (rDNA)的保守区域设计了一套独特的引物。并对这些引物对在禾科植物中的普遍性、特异性和敏感性进行了评价。结论:总的来说,本研究中提出的内参基因集是对滨荆转录组进行qPCR分析的理想归一化基因。应用于本研究的新型内参基因如RPS3比常用的内参基因具有更高的表达稳定性。介绍了rdna引物在qPCR分析中的应用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
5.20
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: Journal of Biological Research-Thessaloniki is a peer-reviewed, open access, international journal that publishes articles providing novel insights into the major fields of biology. Topics covered in Journal of Biological Research-Thessaloniki include, but are not limited to: molecular biology, cytology, genetics, evolutionary biology, morphology, development and differentiation, taxonomy, bioinformatics, physiology, marine biology, behaviour, ecology and conservation.
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