Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas

Darvasi, Ottó; Szabó, Péter M.; Németh, Krisztián; Szabó, Katalin; Spisák, Sándor; Láng, István; Czirják, Sándor; Rácz, Kàroly; Igaz, Péter; Patócs, Attila; Butz, Henriett

doi:10.1007/s12253-017-0330-3

Cited by 11 publications

(8 citation statements)

References 35 publications

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“…al. used the same patient samples for TLDA analyses and analyses using single TaqMan assays, and were only able to validate the TLDA findings for 76.2% of the microRNAs investigated [ 28 ]. In general caution should be taken when comparing even highly similar methods and findings should be validated using identical methods.…”

Section: Discussionmentioning

confidence: 99%

Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions

et al. 2018

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Numerous studies have reported a potential role for circulating microRNAs as biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge some of which might be due to differences in preanalytical and/or analytical factors. Thus, in the current study we systematically investigated the impact of selected preanalytical and analytical variables on the measured microRNA levels in plasma. Similar levels of microRNA were found in platelet-poor plasma obtained by dual compared to prolonged single centrifugation. In contrast, poor correlation was observed between measurements in standard plasma compared to platelet-poor plasma. The correlation between quantitative real-time PCR and droplet digital PCR was found to be good, contrary to TaqMan Low Density Array and single TaqMan assays where no correlation could be demonstrated. Dependent on the specific microRNA measured and the normalization strategy used, the intra- and inter-assay variation of quantitative real-time PCR were found to be 4.2–6.8% and 10.5–31.4%, respectively. Using droplet digital PCR the intra-assay variation was 4.4–20.1%, and the inter-assay variation 5.7–26.7%. Plasma preparation and microRNA purification were found to account for 39–73% of the total intra-assay variation, dependent on the microRNA measured and the normalization strategy used. In conclusion, our study highlighted the importance of reporting comprehensive methodological information when publishing, allowing others to perform validation studies where preanalytical and analytical variables as causes for divergent results can be minimized. Furthermore, if microRNAs are to become routinely used diagnostic or prognostic biomarkers, the differences in plasma microRNA levels between health and diseased subjects must exceed the high preanalytical and analytical variability.

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Section: Discussionmentioning

confidence: 99%

Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions

et al. 2018

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“…The advent of deep sequencing has substantially improved the quality of scientific discovery in many genomic research fields [17][18][19]. Darvasi et al [20] found that the difference among high-throughput platforms is of great importance and selection of screening method can influence experimental results. And miRNA expression profiles could be different using various platforms [21].…”

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confidence: 99%

Next-generation sequencing of microRNAs reveals a unique expression pattern in different types of pituitary adenomas

Chen

et al. 2019

Endocr J

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Pituitary adenomas (PAs) are considered the most common intracranial tumor to cause serious morbidity because of dysregulated pituitary hormone secretions. Aberrant expression of microRNAs (miRNAs) is correlated with the development and function of the pituitary gland as well as the tumorigenesis of hypothalamic-pituitary axis-related pituitary tumors. In this study, we showed the differential expression patterns of miRNAs in NFPAs (nonfunctioning pituitary adenomas), GHPAs (growth hormone-secreting pituitary adenomas) and PRLPAs (prolactin-secreting pituitary adenomas) compared to those in three normal pituitary glands using the HiSeq 2000 sequencing system (Illumina). We validated miRNA expression using real-time quantitative polymerase chain reaction (RT-qPCR) analyses of samples from 73 patients (13 GHPAs, 42 NFPAs, and 18 PRLPAs) and 6 normal pituitary gland. We observed that miR-34c-3p was significantly downregulated in our PRLPA samples (p < 0.01), along with miR-34b-5p, miR-378 and miR-338-5p (all p < 0.05). In NFPAs, miR-493-5p was downregulated, and miR-181b-5p was significantly upregulated (p < 0.01). In GHPAs, miR-184 was significantly upregulated (p < 0.05). We observed that the tumor suppressive miR-124-3p was downregulated in both NFPAs and GHPAs. Taken together, we showed distinctive miRNA expression patterns in these three PAs, and these miRNA signatures in PA may have therapeutic potential as novel biomarkers for each type of PA.

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“…In addition to microarray and RT-qPCR based arrays, next-generation-sequencing (NGS) is the other available high-throughput method for miRNA profiling (miRNome). Compared to microarrays and PCR-based methods, deep-sequencing does not require predesigned probes, thereby allowing for the simultaneous discovery of new miRNAs and the confirmation of known miRNAs [78]. Mutations in miRNAs could also be identified by ultra-deep sequencing, even if these mutations occur in only a small fraction of the sample [79].…”

Section: Discussionmentioning

confidence: 99%

“…The computational tools for analysis are in their infancy [84]. Some NGS library preparation methods and the sequencing technology are not developed for short (<35 bp) sequences [78]. With the rapid increase in miRNAs being discovered and deposited in public databases, NGS can offer another comprehensive view of the miRNA transcriptome and provide a useful complement to microarray assays [84].…”

Section: Discussionmentioning

confidence: 99%

MicroRNA Expression Profiles in the Subcutaneous Adipose Tissues of Morbidly Obese Chinese Women

Wang¹,

Chen²,

Pan³

et al. 2021

Obes Facts

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Introduction: Obesity is a main global health issue and an outstanding cause of morbidity and mortality. Exploring miRNA profiling may help further studies on obesity. Methods: Three morbidly obese and 5 normal-weight Chinese women were enrolled in the microarray testing group. Abdominal subcutaneous adipose tissue (SAT) samples were excised. Total RNAs including miRNAs were extracted. Affymetrix GeneChip miRNA 4.0 Array was used to compare the expression profiles of miRNAs between the 2 groups. Two algorithms, miRanda and TargetScan, were used to predict target messenger RNAs (mRNAs). Bioinformatics analysis was then done based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The sample sizes were further expanded to 8 morbidly obese and 9 normal-weight subjects, and quantitative real-time PCR (qRT-PCR) was utilized to verify the expression of differential miRNAs and target genes. Results: As per the microarray assay, 58 miRNAs were differentially expressed in the SAT from the morbidly obese and normal-weight groups (Fold >4, p < 0.01, FDR <0.05); 54 of these were downregulated and 4 were upregulated in morbidly obese subjects. A total of 1,333 target genes were jointly predicted by miRanda and TargetScan. Further bioinformatics analysis showed that the differential miRNAs were involved in 269 significant biological functions and 89 significant signaling pathways. The validation experiment by qRT-PCR showed that the expression levels of miRNA-143-5p, miRNA-143-3p, miRNA-145-5p, and let-7a-5p were downregulated in morbidly obese subjects, consistent with the microarray detection. High-mobility group A2 (HMGA2), a target gene of the downregulated miRNA let-7a-5p, was first found to be upregulated 3.19-fold in the SAT of morbidly obese Chinese women when compared to normal-weight controls. Conclusions: MiRNA downregulation is a hallmark of intact SAT in a morbidly obese state. Transcription (DNA-dependent), small-molecule metabolic processes, the MAPK signaling pathway, and cancer-related pathways may play important roles in the occurrence and development of obesity. For the first time, we proved that HMGA2, a target gene of let-7a-5p, is upregulated in the SAT of morbidly obese Chinese women.

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Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas

Abstract: 249 words (max: 250)

Cited by 11 publications

References 35 publications

Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions

Quantification of microRNA levels in plasma – Impact of preanalytical and analytical conditions

Next-generation sequencing of microRNAs reveals a unique expression pattern in different types of pituitary adenomas

MicroRNA Expression Profiles in the Subcutaneous Adipose Tissues of Morbidly Obese Chinese Women

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