Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

Zhou, Zhe; Peng, Cong; Tian, Yi; Zhu, Yanmin

doi:10.1371/journal.pone.0185288

Cited by 54 publications

(38 citation statements)

References 47 publications

(46 reference statements)

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“…miR-423/484/92a/b modestly increased early on (~1.15 fold t2 or t3 relative to t1), and further increased ~1.7-fold upon the fourth 24 sampling (t4 / t1), while miR-29/146/148/191 decreased by up to 1.6-fold upon the fourth sampling ( Figure S1A-C). The correlation of miR-423/484/92a/b levels to disease progression 26 was also observed when time from enrolment (in months) was considered as a continuous variable ( Figure S2A-D), while those of miR-29/146/148/191 showed a non-significant 28 correlation (Spearman || < 0.2, p>0.05; not shown) and were not followed up further. Individual trajectories of miR-423/484/92a/b exhibited increases in most of the patients from t1 to t4 ( Figure 30 S3E-H).…”

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confidence: 70%

Circulating miR-181 is a prognostic biomarker for amyotrophic lateral sclerosis

Magen

Coenen-Stass

Großkreutz

et al. 2019

Preprint

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word count: 203 Main text word count: 2738 words (excluding references and figure legends).No. of items (figures, tables): 4, 1 One Sentence Summary: higher plasma miR-181a-5p levels increase mortality risk in ALS patients. Abstract (210 words)Amyotrophic lateral sclerosis (ALS) is a ruthless neurodegenerative disease affecting the motor neuron system. Variability of disease progression has limited the effectiveness of ALS clinical trials, making novel tools for patient stratification at trial recruitment. Thus, better outcome measures are desperately needed in order to achieve therapeutic progress. Here, we investigate the potential of plasma cell-free microRNAs as biomarkers to predict ALS progression. We apply an unbiased high-throughput approach to define miRNA levels in a large cohort of ALS patients and controls. Crucially, we conduct our analysis also on longitudinal samples reflecting disease progression, and are able to integrate detailed clinical phenotyping in our analysis. We identify miR-181a-5p levels to be stable during disease course and demonstrate that high miR-181a-5p plasma levels predict shortened survival in ALS patients, with a 2.5 fold reduction in median survival for patients with high miR-181a-5p plasma levels. We replicated this finding in an independent validation cohort, where an eight-fold (×8) difference in miR-181a-5p levels between the two prognosis subgroups was robustly measurable by quantitative real time PCR. In conclusion, miR-181a-5p plasma levels can predict disease course in ALS suggesting it as a novel biomarker for patient stratification that might will greatly enhance the effectiveness of clinical trials.

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mentioning

confidence: 70%

Circulating miR-181 is a prognostic biomarker for amyotrophic lateral sclerosis

Magen

Coenen-Stass

Großkreutz

et al. 2019

Preprint

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“…This means that we could have potentially excluded unknown and undiscovered stable references. A more thorough approach would be to choose candidate genes from high-throughput data to avoid this inherent bias (24)(25)(26).…”

Section: Discussionmentioning

confidence: 99%

Optimal use of statistical methods to validate reference gene stability in longitudinal studies

Sundaram

Sampathkumar

Massaad

et al. 2019

Preprint

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Multiple statistical approaches have been proposed to validate reference genes in qPCR assays. However, conflicting results from these statistical methods pose a major hurdle in the choice of the best reference genes. Recent studies have proposed the use of a minimum of three different methods but there is no consensus on how to interpret conflicting results. Researchers resort to averaging the ranks or attributing a weighted rank to candidate genes. However, we report here that the suitability of these validation methods can be influenced by the experimental setting. Therefore, averaging the ranks can lead to suboptimal assessment of stable reference genes if the method used is not suitable for analysis. As the respective approaches of these statistical methods are different, a clear understanding of the fundamental assumptions and the parameters that influence the reference gene stability calculation is necessary. In this study, the stability of 10 candidate reference genes (Actb, Gapdh, Tbp, Sdha, Pgk1, Ppia, Rpl13a, Hsp60, Mrpl10, Rps26) was assessed using four common statistical approaches (GeNorm, NormFinder, Coefficient of Variation or CV analysis and Pairwise ΔCt method) in a longitudinal experimental setting. We used the development of the cerebellum and the spinal cord of mice as a model to assess the suitability of these statistical methods for reference gene validation. GeNorm and the Pairwise ΔCt were found to be ill suited due to a fundamental assumption in their stability calculations. Highly correlated genes were given better stability ranks despite significant overall variation. NormFinder fares better but the presence of highly variable genes influences the ranking of all genes because of the algorithm's construct. CV analysis estimates overall variation, but it fails to consider variation across groups. We thus highlight the assumptions and potential pit-falls of each method using our longitudinal data. Based on our results, we have devised a workflow combining NormFinder, CV analysis along with visual representation of mRNA fold changes and one-way ANOVA for validating reference genes in longitudinal studies. This workflow proves to be more robust than any of these methods used individually.

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“…rootstocks. Microarray and transcriptome databases are excellent resources to search for new candidate housekeeping genes, enabling identify several genes with more stable expression than classical reference genes in some plant species [28][29][30][31]. In the present study, 611 candidate housekeeping genes were identified from RNAseq data from our previous work that compared root transcriptomes of two rootstocks 'Mariana 2426' and 'Mazzard F12/1' under hypoxia treatments (S1 Table).…”

Section: Plos Onementioning

confidence: 99%

Identifying and validating housekeeping hybrid Prunus spp. genes for root gene-expression studies

et al. 2020

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Prunus rootstock belonging to subgenera Amygdalus (peach), Prunus (plum) and Cerasus (cherry) are either from the same species as the scion or another one. The number of interspecies (including inter-subgenera) hybrids has increased as a result of broadening the genetic basis for stress (biotic and abiotic) resistance/tolerance. Identifying genes associated with important traits and responses requires expression analysis. Relative quantification is the simplest and most popular alternative, which requires reference genes (housekeeping) to normalize RT-qPCR data. However, there is a scarcity of validated housekeeping genes for hybrid Prunus rootstock species. This research aims to increase the number of housekeeping genes suitable for Prunus rootstock expression analysis. Twenty-one candidate housekeeping genes were pre-selected from previous RNAseq data that compared the response of root transcriptomes of two rootstocks subgenera to hypoxia treatment, 'Mariana 2624' (P. cerasifera Ehrh.× P. munsoniana W. Wight & Hedrick), and 'Mazzard F12/1' (P. avium L.). Representing groups of low, intermediate or high levels of expression, the genes were assayed by RT-qPCR at 72 hours of hypoxia treatment and analyzed with NormFinder software. A sub-set of seven housekeeping genes that presented the highest level of stability were selected, two with low levels of expression (Unknown 3, Unknown 7) and five with medium levels (GTB 1, TUA 3, ATPase P, PRT 6, RP II). The stability of these genes was evaluated under different stress conditions, cold and heat with the hybrid 'Mariana 2624' and N nutrition with the hybrids 'Colt' (P. avium × P. pseudocerasus Lindl.) and 'Garnem' [P. dulcis Mill.× (P. persica L.× P. davidiana Carr.)]. The algorithms of geNorm and BestKeeper software also were used to analyze the performance of these genes as housekeepers.Stability rankings varied according to treatments, genotypes and the software for evaluation, but the gene GBT 1 often had the highest ranking. However, most of the genes are suitable depending on the stressor and/or genotype to be evaluated. No optimal number of reference genes could be determined with geNorm software when all conditions and genotypes were considered. These results strongly suggest that relative RT-qPCR should be PLOS ONE

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Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

Cited by 54 publications

References 47 publications

Circulating miR-181 is a prognostic biomarker for amyotrophic lateral sclerosis

Circulating miR-181 is a prognostic biomarker for amyotrophic lateral sclerosis

Optimal use of statistical methods to validate reference gene stability in longitudinal studies

Identifying and validating housekeeping hybrid Prunus spp. genes for root gene-expression studies

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