Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers

Kok, Maayke G.M.; Ronde, Maurice W J de; Moerland, Perry D.; Ruijter, Jan M.; Creemers, Esther E.; Pinto‐Sietsma, Sara‐Joan

doi:10.1016/j.bdq.2017.11.002

Cited by 83 publications

(68 citation statements)

References 49 publications

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“…We aimed to include a small sample size, 12 horses in total, three of each subgroup, which is commonly used in pilot studies because the method of high‐throughput sequencing is very expensive. This approach will allow us to calculate the necessary sample size for validation of the results in larger study cohorts . Horses were only included in the study on histological confirmation of tumour type.…”

Section: Methodsmentioning

confidence: 99%

“…This approach will allow us to calculate the necessary sample size for validation of the results in larger study cohorts. 25 Horses were only included in the study on histological confirmation of tumour type. The lesions of horses with ES were scored according to a previously established score for clinical disease that ranged from 3 to 19.…”

Section: Study Cohort and Sample Collectionmentioning

confidence: 99%

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Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma

Bogedale

Jagannathan

Gerber

et al. 2019

Vet Comparative Oncology

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The aim of this study was to investigate microRNA (miRNA) differential expression in the two most common equine skin tumours, equine sarcoid (ES) and squamous cell carcinoma (SCC), and its potential influence on the tumour microenvironment at post‐transcriptional level. We investigated miRNA fingerprints in four subgroups: mild (ESM) and aggressive (ESA) ES and ocular SCC (oSCC) and genital SCC (gSCC). Three tumours and three control samples were included in each of the four subgroups. Following next generation sequencing, miRNA differential expression analysis using DESeq2 was carried out. Pathways associated with the human mature homologues of identified dysregulated miRNAs were predicted using DIANA‐ miRPath v3.0. When comparing tumour vs control tissue, 57 miRNAs in ESM, six in ESA, 47 in oSCC and zero in gSCC were found to be differentially expressed and may thus serve as potential diagnostic tissue biomarkers. Whereas, ES lesions in general were associated with downregulation of the miR‐200 family, which may trigger epithelial‐mesenchymal transition, ESM lesions were associated with upregulation of the proposed tumour‐suppressive miRNA cluster on equine chromosome 24. In contrast, the oSCC tumours showed downregulation of this cluster as well as downregulation of the miR‐34 family, which may favour oSCC tumour cell metabolism. To further validate the proposed diagnostic miRNA fingerprints and their suggested biological effects, further miRNA studies need to be carried out in larger study cohorts.

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Section: Methodsmentioning

confidence: 99%

Section: Study Cohort and Sample Collectionmentioning

confidence: 99%

Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma

Bogedale

Jagannathan

Gerber

et al. 2019

Vet Comparative Oncology

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“…Because of the high costs associated with next generation sequencing, a low number of study subjects (5 horses per group) were selected for this exploratory cohort study. Because no general consensus currently exists on what the sample size should be for miRNA biomarker studies, a previously recommended approach was used …”

Section: Methodsmentioning

confidence: 99%

Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression

Unger

Jagannathan

Pacholewska

et al. 2018

Veterinary Internal Medicne

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BackgroundCurrently no methods are available to predict the clinical outcome of individual horses with equine sarcoid (ES) disease.ObjectiveTo investigate if whole blood microRNA (miRNA) profiles can predict the long‐term development of ES tumors.AnimalsFive horses with regression and 5 with progression of ES lesions monitored over 5‐7 years and 5 control horses free of ES for at least 5 years.MethodsFor this cohort study, RNA extracted from whole blood samples from the regression, progression, and control groups was used for high throughput sequencing. Known and novel miRNAs were identified using miRDeep2 and differential expression analysis was carried out by the DESeq2 algorithm. Target gene and pathway prediction as well as enrichment and network analyses were conducted using TarBase, mirPath, and metaCore from GeneGo.ResultsFourteen miRNAs were differentially expressed between regression and progression groups after accounting for the control condition: 4 miRNAs (28.6%) were upregulated and 10 miRNAs (71.4%) were downregulated with >2‐fold change. Seven of the 10 downregulated miRNAs are encoded in an miRNA cluster on equine chromosome 24, homologous to the well‐known 14q32 cluster in humans. Their target genes show enrichment for pathways involved in viral carcinogenesis.Conclusions and Clinical ImportanceWhole blood miRNA expression profiles are associated with long‐term ES growth in horses and warrant further validation as prognostic biomarkers in a larger study cohort. Deregulation of miRNAs on equine chromosome 24 might represent a trigger for ES development.

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“…For example, circulating levels of hsa-miR-145 were lower in women with endometriosis compared to controls [31] whereas hsa-miR-145 levels did not differ [47] or were higher in women with endometriosis compared to the control groups [50]. We postulate that divergent results may be the consequence of different screening platforms and technologies used to identify candidate miRNA markers of disease [57][58][59] and control group characteristics. Moreover, we suggest that different reference material used to quantify circulating miRNA levels are an additional source of variation.…”

Section: Candidate Clinical Markers Of Endometriosismentioning

confidence: 99%

microRNA and Overcoming the Challenges of Their Use in the Diagnosis of Endometriosis

Turpin¹,

Леонова²,

Agarwal³

et al. 2021

Endometriosis

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Endometriosis is a common estrogen dependent and inflammatory disease affecting approximately 176 million women worldwide. Currently, the time between onset of symptoms and a definitive diagnosis has been reported by several international studies to range from 6 to 12 years. Presently, laparoscopic surgery followed by histopathological confirmation of lesions remains the gold standard for diagnosis. In part because of cost and invasiveness, current trends favor reduced laparoscopic surgeries in preference of the non-surgical diagnosis of endometriosis. However, the search for a clinical marker or markers of endometriosis that provide equal or similar sensitivity and specificity to laparoscopy has remained elusive. Thus, the search for a diagnostic test for the diagnosis of endometriosis continues to be a high priority research and clinical issue. Recent studies have reported favorable results with microRNA; however, lack of replication and absence of validation suggest that circulating miRNA may not be reliable for clinical use. Use of different screening platforms together with divergent methods may account for some of the lack or reproducibility in the literature. Herein we critically assess the recent literature and explore sources for discrepant findings. We suggest that prospective studies using validated reference miRNA to normalize results together with improved study design may yet reveal a suitable diagnostic marker or panel of markers for the diagnosis of endometriosis.

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Small sample sizes in high-throughput miRNA screens: A common pitfall for the identification of miRNA biomarkers

Cited by 83 publications

References 49 publications

Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma

Differentially expressed microRNAs, including a large microRNA cluster on chromosome 24, are associated with equine sarcoid and squamous cell carcinoma

Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression

microRNA and Overcoming the Challenges of Their Use in the Diagnosis of Endometriosis

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