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Identification and validation of reference genes for qRT-PCR analysis in mulberry (Morus alba L.).

ABSTRACT: Mulberry (Morus alba L.) is an important economic tree species in many countries. Quantitative real time PCR (qRT-PCR) has become a widely used method for gene expression studies in plants. A suitable reference gene is essential to ensure accurate and reliable results for qRT-PCR analyses. However, no reports describing the selection of reference genes have been published for mulberry. In this work, we evaluated the stability of twenty candidate reference genes in different plant tissues and under different stress conditions by qRT-PCR in mulberry using algorithms in two programs-geNorm and NormFinder. The results revealed that TUB2, UBI4, ACTIN3 and RPL4 were ranked as the most stable reference genes in the samples subsets, whereas EF1?4 and TUB3showed the least stability with both algorithms. To further validate the stability of the reference genes, the expression patterns of six genes of mulberry were analyzed by normalization with the selected reference genes. Our study will benefit future analyses of gene expression in mulberry.

SUBMITTER: Dai F

PROVIDER: S-EPMC5854264 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Identification and validation of reference genes for qRT-PCR analysis in mulberry (Morus alba L.).

Dai Fanwei F Zhao Xiting X Tang Cuiming C Wang Zhenjiang Z Kuang Zheshi Z Li Zhiyi Z Huang Jing J Luo Guoqing G

PloS one 20180315 3

Mulberry (Morus alba L.) is an important economic tree species in many countries. Quantitative real time PCR (qRT-PCR) has become a widely used method for gene expression studies in plants. A suitable reference gene is essential to ensure accurate and reliable results for qRT-PCR analyses. However, no reports describing the selection of reference genes have been published for mulberry. In this work, we evaluated the stability of twenty candidate reference genes in different plant tissues and und ...[more]

PMID: 29543877

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Project description:BackgroundBivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and ?-actin (ACT) is most frequently used as qRT-PCR reference gene without validation.ResultsIn this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopectenyessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ?Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.ConclusionsThis work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

Project description:Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the C(T) values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ?C(T) method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species.

Dataset Information

Identification and validation of reference genes for qRT-PCR analysis in mulberry (Morus alba L.).

Publications

Identification and validation of reference genes for qRT-PCR analysis in mulberry (Morus alba L.).

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