Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

Prado, Marcelle SanJuan Ganem; Goes, Thaline Cunha de; Jesus, Mirthz Lemos de; Mendonça, Lucilla Silva Oliveira; Nascimento, Jadson Santos; Kaneto, Carla Martins

doi:10.1038/s41598-019-56172-w

Cited by 13 publications

(15 citation statements)

References 38 publications

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“…The results from RT-qPCR expression (CTs) were normalized using the endogenous and most stable miRNA in plasma or tissue analyses, selected using the RefFinder software [ 14 ] following previously stated strategies [ 66 , 67 ]. This software comprises the results of 4 commonly employed algorithms: Delta CT, BestKeeper, Normfinder, and Genorm.…”

Section: Methodsmentioning

confidence: 99%

Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients

Plana

Gálvez

Medina

et al. 2020

IJMS

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microRNAs (miRNAs) are small RNAs that regulate different biological processes. Our objective was to identify miRNAs dysregulated in plasma and tissue of patients with abdominal aortic aneurysm (AAA) and explore new potential targets involved in AAA. Fifty-seven subjects were recruited for a plasma study (30 AAA patients, 16 healthy volunteers and 11 patients with atherosclerosis). The expression level of 179 miRNAs was screened in plasma from a subset of samples, and dysregulated miRNAs were validated in the entire study population. Dysregulated miRNAs were also quantified in aortic tissue of 21 AAA patients and 8 organ donors. Applying a gene set enrichment analysis, an interaction map of dysregulated miRNAs and their targets was built, and selected targets were quantified in tissue samples. miR-27b-3p and miR-221-3p were overexpressed in plasma of AAA patients compared with healthy controls, 1.6 times and 1.9 times, respectively. In AAA tissue, six miRNAs (miR-1, miR-27b-3p, miR-29b-3p, miR-133a-3p, miR-133b, and miR-195-5p) were underexpressed from 1.6 to 4.8 times and four miRNAs (miR-146a-5p, miR-21-5p, miR-144-3p, and miR-103a-3p) were overexpressed from 1.3 to 7.2 times. Thrombospondin-2, a target of miR-195-5p, was increased in AAA tissue and negatively correlated with the expression of miR-195-5p, suggesting their involvement in a common regulatory mechanism.

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Section: Methodsmentioning

confidence: 99%

Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients

Plana

Gálvez

Medina

et al. 2020

IJMS

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“…There is no consensus for the best reference gene to be used to normalize the qRT-PCR data in biological fluid samples. Initially, external synthetic miRNA molecules called spike-ins were used for normalization, but in recent times, identifying and using a specific endogenous miRNA as a reference are suggested ( 14 – 17 ). The best way to normalize the plasma miRNAs is to use an endogenous host miRNA, which is stable, abundant, and detectable without any significant variation in its expression status across the various sample groups.…”

Section: Resultsmentioning

confidence: 99%

High-Throughput RNA Sequencing Analysis of Plasma Samples Reveals Circulating microRNA Signatures with Biomarker Potential in Dengue Disease Progression

Saini

Bandyopadhyay²,

Pandey

et al. 2020

mSystems

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The circulating microRNA (miRNA) profile has been widely used for identifying potential biomarkers against viral infections. However, data on circulating microRNA expression patterns in dengue patients are scanty. Considering the impact of severity caused by dengue infection, circulating miRNA profiles in plasma of dengue patients may prove to be valuable for developing early prognostic markers for the disease severity. Here, we described an in-depth analytical study of small RNA sequencing data obtained from the plasma of 39 dengue patients. Integrating bioinformatics and in vitro studies, we identified differentially expressed miRNAs (DEMs) (log2 fold change ≥1.5, P < 0.05) associated with dengue disease progression. In comparing miRNA expression pattern with the follow-up samples, nine miRNAs were found to exhibit an altered expression that could distinguish between severe dengue and the convalescent patients. To understand the abundance and specificity of the DEMs in the context of dengue infection and disease progression, eight top-hit DEMs were further validated in the dengue virus-infected cell lines as well as in the patient’s plasma and peripheral blood mononuclear cells (PBMCs) using the quantitative reverse transcription-PCR (qRT‐PCR) method. Importantly, receiver operating curve analysis further confirmed that the plasma expression pattern of hsa-miR-122-5p could differentiate between different stages of dengue infection (area under the concentration-time curve [AUC] = 0.792), and dengue-negative patients with other febrile illnesses (AUC = 0.984). The in silico analysis of DEM target genes suggested an enrichment of the pathways associated with metabolism and inflammation. Our study gives a global view of miRNA expression in the plasma from dengue patients and provides a precious resource of candidate miRNAs involved in dengue infection and disease progression. IMPORTANCE Dengue virus (DENV) infection usually causes dengue fever (DF) with flu-like illness affecting infants, young children, and adults. The DF occasionally evolves into a potentially lethal complication called dengue severe (DS) leading to a rapid fall in platelet count along with plasma leakage, fluid accumulation, respiratory distress, and severe bleeding. The diverse clinical spectrum of dengue disease, as well as its significant similarity to other febrile viral illnesses, makes early identification more challenging in this high-risk group. microRNAs (miRNAs) are small (∼19 to 21 nucleotides [nt] in length), noncoding RNAs, extremely stable and easily detectable in the plasma; thus, they have potential as biomarkers for diagnosing and monitoring human diseases. This study provides a comprehensive analysis of miRNAs circulating in plasma of dengue virus-infected patients and identifies the miRNA signatures that have biomarker potential for dengue infection and disease progression.

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“…Several molecules have been proposed as normalizers for miRNAs studies in different disorders, mainly in blood [ 36 ], plasma [ 37 , 38 , 39 ], cell cultures studies [ 25 , 40 ], tissue [ 41 ] and urine [ 35 , 42 , 43 , 44 ]. However, there is no consensus, even in the same sample type, regarding which is the best normalizer for miRNA studies.…”

Section: Discussionmentioning

confidence: 99%

Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer

et al. 2020

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Bladder cancer (BC) is among the most frequent malignancies worldwide, being the most expensive cancer to treat and monitor and the most lethal urological cancer. Urine microRNAs (miRNAs) have been proposed as novel non-invasive biomarkers to early diagnose and monitor BC patients in order to avoid the performance of current aggressive diagnostic techniques. However, huge discrepancies arise among studies mainly due to the lack of standardization in the normalization, a crucial step in all miRNA studies. Our aim was to identify the best miRNA normalizer for miRNA studies in urine of BC patients. We evaluated the performance of 110 candidate miRNAs in urine of 35 BC patients and 15 healthy controls by Real Time quantitative Polymerase Chain Reaction (RT-qPCR) followed by a stability analysis with RefFinder. In this screening stage, miR-29c-3p arose as the most stably expressed miRNA in BC and controls, with a good expression level. Stability of miR-29c-3p expression was validated in an independent cohort of 153 BC patients and 57 controls. Finally, we evaluated the robustness of miR-29c-3p as normalizer in the expression study of miR-200c-3p, a potential diagnostic marker for BC. We propose miR-29c-3p as a normalizer for miRNA studies in BC urine. This is the first study that characterizes a reliable normalizer that may allow the comparison of future urine miRNA studies as non-invasive biomarkers for BC diagnosis and monitoring.

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Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

Cited by 13 publications

References 38 publications

Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients

Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients

High-Throughput RNA Sequencing Analysis of Plasma Samples Reveals Circulating microRNA Signatures with Biomarker Potential in Dengue Disease Progression

Identification of miR-29c-3p as a Robust Normalizer for Urine microRNA Studies in Bladder Cancer

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