Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri

Lyu, Shiheng; Yu, Ying; Xu, Shirong; Cai, Weiwei; Chen, Guixin; Chen, Jianjun; Dong-ming, Pan; She, Wenqin

doi:10.3390/genes11010017

Cited by 10 publications

(5 citation statements)

References 120 publications

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“…Therefore, its use as a reference would not be optimal for use when comparing changes in gene expression associated with induction of differentiation over time [35]. This finding has been reported from studies of differentiation of human adipose-derived mesenchymal stem cells [36], from human liver samples [37], in non-alcoholic fatty liver disease (NAFLD) animal models [38], different pig tissues [39], and plant samples [40]. Those reports show that when GAPDH was used as an internal control, the investigated GOI had no significant change or had fluctuating patterns of expression, indicating that GAPDH is unreliable for RT-qPCR analysis.…”

Section: Discussionmentioning

confidence: 97%

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Brzeszczyńska

Brzeszczyński

Samuel

et al. 2020

Cells

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Gene expression analysis by quantitative real-time polymerase chain reaction (RT-qPCR) is routinely used in biomedical studies. The reproducibility and reliability of the data fundamentally depends on experimental design and data interpretation. Despite the wide application of this assay, there is significant variation in the validation process of gene expression data from research laboratories. Since the validity of results depends on appropriate normalisation, it is crucial to select appropriate reference gene(s), where transcription of the selected gene is unaffected by experimental setting. In this study we have applied geNorm technology to investigate the transcription of 12 'housekeeping' genes for use in the normalisation of RT-qPCR data acquired using a widely accepted HepaRG hepatic cell line in studies examining models of pre-clinical drug testing. geNorm data identified a number of genes unaffected by specific drug treatments and showed that different genes remained invariant in response to different drug treatments, whereas the transcription of 'classical' reference genes such as GAPDH (glyceralde-hyde-3-phosphate dehydrogenase) was altered by drug treatment. Comparing data normalised using the reference genes identified by geNorm with normalisation using classical housekeeping genes demonstrated substantial differences in the final results. In light of cell therapy application, RT-qPCR analyses has to be carefully evaluated to accurately interpret data obtained from dynamic cellular models undergoing sequential stages of phenotypic change.

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Section: Discussionmentioning

confidence: 97%

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Brzeszczyńska

Brzeszczyński

Samuel

et al. 2020

Cells

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“…To quantify the identified miRNAs and mRNAs, poly(A) extension RT-qPCR was performed using a protocol with minor modifications [79]. All the RT-qPCR templates were generated from 3 µg total RNA isolated from the WT and MT juice sacs at 76, 90, 104 and 118 DAF.…”

Section: Validation Of Mrna and Mirnas Expressions By Quantitative Real-time Pcr Analysismentioning

confidence: 99%

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck)

Pan

Lyu

Chen

et al. 2021

IJMS

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‘Liuyuezaoyou’ is an early-ripening cultivar selected from a bud mutation of Citrus grandis Osbeck ‘Guanximiyou’. They were designated here as MT and WT, respectively. The fruit of MT matures about 45 days earlier than WT, which was accompanied by significant changes in key phytohormones, sugar compounds and organic acids. Recent studies have showed that microRNAs (miRNAs) play an important role in regulation of fruit ripening process. The aim of this study was to compare MT fruits with WT ones to uncover if miRNAs were implicated in the ripening of C. grandis. Fruits of both WT and MT at four developmental stages were analyzed using high-throughput sequencing and RT-PCR. Several independent miRNA libraries were constructed and sequenced. A total of 747 known miRNAs were identified and 99 novel miRNAs were predicted across all libraries. The novel miRNAs were found to have hairpin structures and possess star sequences. These results showed that transcriptome and miRNAs are substantially involved in a complex and comprehensive network in regulation of fruit ripening of this species. Further analysis of the network model revealed intricate interactions of miRNAs with mRNAs during the fleshy fruit ripening process. Several identified miRNAs have potential targets. These include auxin-responsive protein IAA9, sucrose synthase 3, V-type proton ATPase, NCED1 (ABA biosynthesis) and PL1/5 (pectate lyase genes), as well as NAC100 putative coordinated regulation networks, whose interactions with respective miRNAs may contribute significantly to fruit ripening of C. grandis.

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“…In Juglans regia , the most suitable reference miRNAs for flower buds at different differentiation stages were jre-miR394a , jre-miR159a , and jre-miR159c , and the most suitable reference miRNAs for leaf buds at different differentiation stages were 5.8S rRNA and jre-miRn3 [ 37 ]. The most stable reference miRNA combinations during seed development in Brassica napus were miR167-1_2 , miR11-1 , and miR159-1 [ 38 ].…”

Section: Discussionmentioning

confidence: 99%

Screening of Reference miRNA of Different Early- and Late-Flowering Tree Peony Varieties

Shen

Wang²,

et al. 2023

Plants

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miRNA plays an important role in plant growth and development and in response to various stresses. Quantitative real-time PCR (qRT-PCR) technology is often used to detect the expression level of miRNAs and genes by comparing with reference genes. In order to screen out the optimal reference miRNAs in different tree peony varieties, the petals of 42 different early- and late-flowering tree peony varieties were used as experimental materials, and geNorm, NormFinder, Bestkeeper, and RefFinder software were used to evaluate the stability of 16 candidate reference miRNAs. The results showed that the average Ct values of all candidate reference miRNAs were between 15.34 ± 0.29 and 32.64 ± 0.38. The optimal number of reference miRNAs was four, which were PsPC-5p-19095, PsPC-3p-51259, PsmiR159a, and PsPC-3p-6660 in geNorm. The stability of PsPC-3p-6660 was the highest in the analysis results of NormFinder software. Among the analysis results of Bestkeeper software, PsMIR319-p5 has the highest stability. Among the results of comprehensive evaluation and analysis of several software using RefFinder, the candidate reference miRNA with the highest stability was PsPC-3p-6660. When PsPC-3p-6660 was used as the reference miRNA, the expression of PomiR171 and PomiR414 in response to different flowering times of tree peony was relatively stable in 42 tree peony varieties, indicating that PsPC-3p-6660 was stable and reliable. The results of this study provide a reference miRNA for studying the expression changes of miRNA in different tree peony varieties and further exploring the regulatory mechanism of miRNA in different peony varieties.

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Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri

Cited by 10 publications

References 120 publications

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

Validation of Reference Genes for Gene Expression Studies by RT-qPCR in HepaRG Cells during Toxicity Testing and Disease Modelling

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck)

Screening of Reference miRNA of Different Early- and Late-Flowering Tree Peony Varieties

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