Dataset Information

Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita.

ABSTRACT: Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and geNorm suggested the optimal number of reference genes for normalization. RPL13a and RPL18 were the most suitable reference genes for developmental stages, tissues, and temperature treatments; RPL13a and RPS3 were the most suitable for pesticide and photoperiod treatments; RPS18 and RPL18 were the most suitable for the two sexes. We validated the normalized results using odorant-binding protein genes as target genes in different tissues. Compared with the selected suitable reference genes, the expression of OBP1 in antennae, abdomen, and wings, and OBP2 in antennae and wings were overestimated due to the instability of ACTb. These results identified several reliable reference genes in H. oblita for normalization, and are valuable for future molecular studies.

SUBMITTER: Xie M

PROVIDER: S-EPMC7577503 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita.

Xie Minghui M Zhong Yongzhi Y Lin Lulu L Zhang Guangling G Su Weihua W Ni Wanli W Qu Mingjing M Chen Haoliang H

PloS one 20201021 10

Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and g ...[more]

PMID: 33085726

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Project description:BackgroundThe 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.ResultsTen 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.ConclusionsOverall, 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.

Project description:BackgroundReal-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.ResultsA systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.ConclusionNone of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.

Dataset Information

Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita.

Publications

Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita.

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