PARP2 poly(ADP-ribosyl)ates nuclear factor erythroid 2-related factor 2 (NRF2) affecting NRF2 subcellular localization.

PARP2 is a member of the PARP enzyme family. Although, PARP2 plays role in DNA repair, it has regulatory roles in mitochondrial and lipid metabolism, it has pivotal role in bringing about the adverse effects of pharmacological PARP inhibitors. Previously, we showed that the ablation of PARP2 induces oxidative stress and, consequently, mitochondrial fragmentation. In attempt to identify the source of the reactive species we assessed the possible role of a central regulator of cellular antioxidant defense, nuclear factor erythroid 2-related factor 2 (NRF2). The silencing of PARP2 did not alter either the mRNA or the protein expression of NRF2, but changed its subcellular localization, decreasing the proportion of nuclear, active fraction of NRF2. Pharmacological inhibition of PARP2 partially restored the normal localization pattern of NRF2 and in line with that, we showed that NRF2 is PARylated that is absent in the cells in which PARP2 was silenced. Apparently, the PARylation of NRF2 by PARP2 has pivotal role in regulating the subcellular (nuclear) localization of NRF2. The silencing of PARP2 rearranged the expression of genes encoding proteins with antioxidant function, among these a subset of NRF2-dependent genes.

The Role of ARID1A in the Nonestrogenic Modulation of IGF-1 Signaling.

Gaining pharmacologic access to the potential of ARID1A, a tumor suppressor protein, to mediate transcriptional control over cancer gene expression is an unresolved challenge. Retinoid X receptor ligands are pleiotropic, incompletely understood tools that regulate breast epithelial cell proliferation and differentiation. We found that low-dose bexarotene (Bex) combined with the nonselective beta-blocker carvedilol (Carv) reduces proliferation of MCF10DCIS.com cells and markedly suppresses ARID1A levels. Similarly, Carv synergized with Bex in MCF-7 cells to suppress cell growth. Chromatin immunoprecipitation sequencing analysis revealed that under nonestrogenic conditions Bex + Carv alters the concerted genomic distribution of the chromatin remodeler ARID1A and acetylated histone H3K27, at sites related to insulin-like growth factor (IGF) signaling. Several distinct sites of ARID1A enrichment were identified in the IGF-1 receptor and IRS1 genes, associated with a suppression of both proteins. The knock-down of ARID1A increased IGF-1R levels, prevented IGF-1R and IRS1 suppression upon Bex + Carv, and stimulated proliferation. In vitro IGF-1 receptor neutralizing antibody suppressed cell growth, while elevated IGF-1R or IRS1 expression was associated with poor survival of patients with ER-negative breast cancer. Our study demonstrates direct impact of ARID1A redistribution on the expression and growth regulation of IGF-1-related genes, induced by repurposed clinical drugs under nonestrogenic conditions. IMPLICATIONS: This study underscores the possibility of the pharmacologic modulation of the ARID1A factor to downregulate protumorigenic IGF-1 activity in patients with postmenopausal breast cancer undergoing aromatase inhibitor treatment.

Kynurenic Acid and Its Synthetic Derivatives Protect Against Sepsis-Associated Neutrophil Activation and Brain Mitochondrial Dysfunction in Rats.

Background and Aims: The systemic host response in sepsis is frequently accompanied by central nervous system (CNS) dysfunction. Evidence suggests that excessive formation of neutrophil extracellular traps (NETs) can increase the permeability of the blood-brain barrier (BBB) and that the evolving mitochondrial damage may contribute to the pathogenesis of sepsis-associated encephalopathy. Kynurenic acid (KYNA), a metabolite of tryptophan catabolism, exerts pleiotropic cell-protective effects under pro-inflammatory conditions. Our aim was to investigate whether exogenous KYNA or its synthetic analogues SZR-72 and SZR-104 affect BBB permeability secondary to NET formation and influence cerebral mitochondrial disturbances in a clinically relevant rodent model of intraabdominal sepsis. Methods: Sprague-Dawley rats were subjected to fecal peritonitis (0.6 g kg-1 ip) or a sham operation. Septic animals were treated with saline or KYNA, SZR-72 or SZR-104 (160 µmol kg-1 each ip) 16h and 22h after induction. Invasive monitoring was performed on anesthetized animals to evaluate respiratory, cardiovascular, renal, hepatic and metabolic parameters to calculate rat organ failure assessment (ROFA) scores. NET components (citrullinated histone H3 (CitH3); myeloperoxidase (MPO)) and the NET inducer IL-1ß, as well as IL-6 and a brain injury marker (S100B) were detected from plasma samples. After 24h, leukocyte infiltration (tissue MPO) and mitochondrial complex I- and II-linked (CI-CII) oxidative phosphorylation (OXPHOS) were evaluated. In a separate series, Evans Blue extravasation and the edema index were used to assess BBB permeability in the same regions. Results: Sepsis was characterized by significantly elevated ROFA scores, while the increased BBB permeability and plasma S100B levels demonstrated brain damage. Plasma levels of CitH3, MPO and IL-1ß were elevated in sepsis but were ameliorated by KYNA and its synthetic analogues. The sepsis-induced deterioration in tissue CI-CII-linked OXPHOS and BBB parameters as well as the increase in tissue MPO content were positively affected by KYNA/KYNA analogues. Conclusion: This study is the first to report that KYNA and KYNA analogues are potential neuroprotective agents in experimental sepsis. The proposed mechanistic steps involve reduced peripheral NET formation, lowered BBB permeability changes and alleviation of mitochondrial dysfunction in the CNS.

Silencing of Poly(ADP-Ribose) Polymerase-2 Induces Mitochondrial Reactive Species Production and Mitochondrial Fragmentation.

PARP2 is a DNA repair protein. The deletion of PARP2 induces mitochondrial biogenesis and mitochondrial activity by increasing NAD+ levels and inducing SIRT1 activity. We show that the silencing of PARP2 causes mitochondrial fragmentation in myoblasts. We assessed multiple pathways that can lead to mitochondrial fragmentation and ruled out the involvement of mitophagy, the fusion-fission machinery, SIRT1, and mitochondrial unfolded protein response. Nevertheless, mitochondrial fragmentation was reversed by treatment with strong reductants, such as reduced glutathione (GSH), N-acetyl-cysteine (NAC), and a mitochondria-specific antioxidant MitoTEMPO. The effect of MitoTEMPO on mitochondrial morphology indicates the production of reactive oxygen species of mitochondrial origin. Elimination of reactive oxygen species reversed mitochondrial fragmentation in PARP2-silenced cells.

Corrigendum: Investigation of the Possible Role of Tie2 Pathway and TEK Gene in Asthma and Allergic Conjunctivitis.

[This corrects the article DOI: 10.3389/fgene.2020.00128.].

Fat Distribution in Women Is Associated With Depot-Specific Transcriptomic Signatures and Chromatin Structure.

BACKGROUND: Preferential accumulation of fat in the upper body (apple shape) is associated with higher risk of developing metabolic syndrome relative to lower body fat (pear shape). We previously discovered that chromatin openness partially defined the transcriptome of preadipocytes isolated from abdominal and gluteofemoral fat. However, the molecular mechanisms underlying interindividual variation in body shape are unknown. METHODS: Adipocyte fraction was isolated from abdominal and gluteofemoral fat biopsies of premenopausal women (age and body mass index matched) segregated initially only by their waist-to-hip ratio. We evaluated transcriptomic and chromatin accessibility using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) along with key clinical parameters. RESULTS: Our data showed that higher lower body fat mass was associated with better lipid profile and free fatty acid decrease after glucose administration. Lipid and glucose metabolic pathways genes were expressed at higher levels in gluteofemoral adipocyte fraction in pears, whereas genes associated with inflammation were higher both in abdominal and gluteofemoral apple adipocyte fraction. Gluteofemoral adipocyte chromatin from pear-shaped women contained a significantly higher number of differentially open ATAC-seq peaks relative to chromatin from the apple-shaped gluteofemoral adipocytes. In contrast, abdominal adipocyte chromatin openness showed few differences between apple- and pear-shaped women. We revealed a correlation between gene transcription and open chromatin at the proximity of the transcriptional start site of some of the differentially expressed genes. CONCLUSIONS: Integration of data from all 3 approaches suggests that chromatin openness partially governs the transcriptome of gluteofemoral adipocytes and may be involved in the early metabolic syndrome predisposition associated with body shape.

Investigation of the Possible Role of Tie2 Pathway and TEK Gene in Asthma and Allergic Conjunctivitis.

Tie2, coded by the TEK gene, is a tyrosine kinase receptor and plays a central role in vascular stability. It was suggested that variations in the TEK gene might influence the susceptibility to asthma and allergic conjunctivitis. The aim of this study was to further investigate these suggestions, involving different populations and to study the Tie2 related pathway on a mouse model of asthma. The discovery, stage I cohort involved 306 patients with moderate and severe allergic rhinitis, the stage II study consisted of four cohorts, namely, adult and pediatric asthmatics and corresponding controls. Altogether, there were 1,258 unrelated individuals in these cohorts, out of which 63.9% were children and 36.1% were adults. In stage I, 112 SNPs were screened in the TEK gene of the patients in order to search for associations with asthma and allergic conjunctivitis. The top associated SNPs were selected for association studies on the replication cohorts. The rs3824410 SNP was nominally associated with a reduced risk of asthma in the stage I cohort and with severe asthma within the asthmatic population (p=0.009; OR=0.48) in the replication cohort. In the stage I study, 5 SNPs were selected in conjunctivitis. Due to the low number of adult patients with conjunctivitis, only children were involved in stage II. Within the asthmatic children, the rs622232 SNP was associated with conjunctivitis in boys in the dominant model (p=0.004; OR=4.76), while the rs7034505 showed association to conjunctivitis in girls (p=0.012; OR=2.42). In the lung of a mouse model of asthma, expression changes of 10 Tie2 pathway-related genes were evaluated at three points in time. Eighty percent of the selected genes showed significant changes in their expressions at least at one time point during the process, leading from sensitization to allergic airway inflammation. The expressions of both the Tek gene and its ligands showed a reduced level at all time points. In conclusion, our results provide additional proof that the Tie2 pathway, the TEK gene and its variations might have a role in asthma and allergic conjunctivitis. The gene and its associated pathways can be potential therapeutic targets in both diseases.

Altered microRNAs expression levels of sperm and seminal plasma in patients with infertile ejaculates compared with normozoospermic males.

Approximately 15% of couples are unable to conceive after one year of unprotected intercourse. Because sperm can be accessed with ease, it is reasonable to search for non-invasive biomarkers in semen. MicroRNAs are a family of short single-stranded non-coding RNA molecules that are capable of regulating gene expression and causing mRNA degradation. We studied the most common 11 spermatogenesis-related microRNAs expression levels in sperm and seminal plasma from patients with oligozoospermic or asthenozoospermic ejaculates, and in men with normozoospermic ejaculates. Five of these miRNAs were significantly upregulated and three were downregulated in infertile males compared to men with normozoospermic ejaculates. A statistically significant negative correlation was found between the sperm concentration and several microRNA expression level (let-7a, miR-7-1-3p, miR-141, miR-200a, and miR-429, p < 0.0001) both in sperm and in seminal plasma. We also found positive correlation between sperm concentration and some miRNA expression levels (miR-15b, miR-34b, and miR-122, p < 0.001) in sperm and in seminal plasma. This is the first study to demonstrate differences between sperm and seminal plasma miRNA expression level and to identify a correlation between the sperm concentration and miRNAs expression level. Therefore, these MiRNAs could have the potential be used as non-invasive biomarkers to diagnose males with impaired sperm production.

Development of an antibody control system using phage display.

While chromatin immunoprecipitation has become a widely-used method in the field of transcription regulation studies, serious limitations connected to the complexity and relatively little standardization of the method serve as obstacles for its use in clinical research. In this paper we introduce a method for developing bacteriophage-based controls for the better standardization of the chromatin immunoprecipitation reactions. Random phage display libraries were selected with ChIP-grade antibodies for several rounds and individual monoclonal phages were isolated. These monoclonal phages can be propagated, characterized, capillary sequenced and if needed later cloned from in-silico data. Using such control tools allows for a better characterization of the immunoprecipitation stage needed for further clinical research in the field of chromatin-immunoprecipitation-based studies.

Extensive epigenetic and transcriptomic variability between genetically identical human B-lymphoblastoid cells with implications in pharmacogenomics research.

Genotyped human B-lymphoblastoid cell lines (LCLs) are widely used models in mapping quantitative trait loci for chromatin features, gene expression, and drug response. The extent of genotype-independent functional genomic variability of the LCL model, although largely overlooked, may inform association study design. In this study, we use flow cytometry, chromatin immunoprecipitation sequencing and mRNA sequencing to study surface marker patterns, quantify genome-wide chromatin changes (H3K27ac) and transcriptome variability, respectively, among five isogenic LCLs derived from the same individual. Most of the studied LCLs were non-monoclonal and had mature B cell phenotypes. Strikingly, nearly one-fourth of active gene regulatory regions showed significantly variable H3K27ac levels, especially enhancers, among which several were classified as clustered enhancers. Large, contiguous genomic regions showed signs of coordinated activity change. Regulatory differences were mirrored by mRNA expression changes, preferentially affecting hundreds of genes involved in specialized cellular processes including immune and drug response pathways. Differential expression of DPYD, an enzyme involved in 5-fluorouracil (5-FU) catabolism, was associated with variable LCL growth inhibition mediated by 5-FU. The extent of genotype-independent functional genomic variability might highlight the need to revisit study design strategies for LCLs in pharmacogenomics.

Differential open chromatin profile and transcriptomic signature define depot-specific human subcutaneous preadipocytes: primary outcomes.

BACKGROUND: Increased lower body fat is associated with reduced cardiometabolic risk. The molecular basis for depot-specific differences in gluteofemoral (GF) compared with abdominal (A) subcutaneous adipocyte function is poorly understood. In the current report, we used a combination of Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq), RNA-seq, and chromatin immunoprecipitation (ChIP)-qPCR analyses that provide evidence that depot-specific gene expression patterns are associated with differential epigenetic chromatin signatures. METHODS: Preadipocytes cultured from A and GF adipose tissue obtained from premenopausal apple-shaped women were used to perform transcriptome analysis by RNA-seq and assess accessible chromatin regions by ATAC-seq. We measured mRNA expression and performed ChIP-qPCR experiments for histone modifications of active (H3K4me3) and repressed chromatin (H3K27me3) regions respectively on the promoter regions of differentially expressed genes. RESULTS: RNA-seq experiments revealed an A-fat and GF-fat selective gene expression signature, with 126 genes upregulated in abdominal preadipocytes and 90 genes upregulated in GF cells. ATAC-seq identified almost 10-times more A-specific chromatin-accessible regions. Using a combined analysis of ATAC-seq and global gene expression data, we identified 74 of the 126 abdominal-specific genes (59%) with A-specific accessible chromatin sites within 200 kb of the transcription start site (TSS), including HOXA3, HOXA5, IL8, IL1b, and IL6. Interestingly, only 14 of the 90 GF-specific genes (15%) had GF-specific accessible chromatin sites within 200 kb of the corresponding TSS, including HOXC13 and HOTAIR, whereas 25 of them (28%) had abdominal-specific accessible chromatin sites. ChIP-qPCR experiments confirmed that the active H3K4me3 chromatin mark was significantly enriched at the promoter regions of HOXA5 and HOXA3 genes in abdominal preadipocytes, while H3K27me3 was less abundant relative to chromatin from GF. This is consistent with their A-fat specific gene expression pattern. Conversely, analysis of the promoter regions of the GF specific HOTAIR and HOXC13 genes exhibited high H3K4me3 and low H3K27me3 levels in GF chromatin compared to A chromatin. CONCLUSIONS: Global transcriptome and open chromatin analyses of depot-specific preadipocytes identified their gene expression signature and differential open chromatin profile. Interestingly, A-fat-specific open chromatin regions can be observed in the proximity of GF-fat genes, but not vice versa. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01745471 . Registered 5 December 2012.

The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory.

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Poly(ADP-ribose) polymerase-2 is a lipid-modulated modulator of muscular lipid homeostasis.

There is a growing body of evidence that poly(ADP-ribose) polymerase-2 (PARP2), although originally described as a DNA repair protein, has a widespread role as a metabolic regulator. We show that the ablation of PARP2 induced characteristic changes in the lipidome. The silencing of PARP2 induced the expression of sterol regulatory element-binding protein-1 and -2 and initiated de novo cholesterol biosynthesis in skeletal muscle. Increased muscular cholesterol was shunted to muscular biosynthesis of dihydrotestosterone, an anabolic steroid. Thus, skeletal muscle fibers in PARP2-/- mice were stronger compared to those of their wild-type littermates. In addition, we detected changes in the dynamics of the cell membrane, suggesting that lipidome changes also affect the biophysical characteristics of the cell membrane. In in silico and wet chemistry studies, we identified lipid species that can decrease the expression of PARP2 and potentially phenocopy the genetic abruption of PARP2, including artificial steroids. In view of these observations, we propose a new role for PARP2 as a lipid-modulated regulator of lipid metabolism.

From genomes to diaries: a 3-year prospective, real-life study of ragweed-specific sublingual immunotherapy.

During the last decades, the prevalence of allergy has dramatically increased. Allergen-specific immunotherapy is the only currently available medical intervention that has the potential to affect the natural course of the disease, but there are still many questions and unmet needs hindering its widespread use to fulfill its treatment potential and maximize its benefits for the society. To provide a comprehensive phenome-wide overview in sublingual immunotherapy, using ragweed allergy as a target, we planned and carried out a longitudinal, prospective, observational, open-label study (DesensIT). In this paper we present challenges of using deep and comprehensive phenotypes embracing biological, clinical and patient-reported outcomes in allergen-specific immunotherapy and show how we designed the DesensIT project to optimize data collection, processing and evaluation.

The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2.

Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression.

Inducible super-enhancers are organized based on canonical signal-specific transcription factor binding elements.

Super-enhancers are established through the interactions of several enhancers and a large number of proteins, including transcription factors and co-regulators; however, the formation of these interactions is poorly understood. By re-analysing previously published estrogen receptor alpha (ERα) ChIP-seq data sets derived from the MCF-7 cell line, we observed that in the absence of stimulation, future super-enhancers are represented by one or a few transcription factor binding event(s) and these extraordinary enhancers possess a response element largely specific to the ERα dimer. Upon hormonal stimulation, these primary binding sites are surrounded by a large amount of ERα and the critical components of active enhancers, such as P300 and MED1, and together with neighbouring sites bound by newly recruited ERα, they generate the functional super-enhancers. To further validate the role of canonical elements in super-enhancer formation, we investigated some additional signal-dependent transcription factors, confirming that certain, distinguished binding elements have a general organizer function. These results suggest that certain signal-specific transcription factors guide super-enhancer formation upon binding to strong response elements. These findings may reshape the current understanding of how these regulatory units assemble, highlighting the involvement of DNA elements instead of protein-protein interactions.

Nuclear receptors in transgenerational epigenetic inheritance.

Nuclear Receptors are ligand-activated transcription factors that translate information about the lipid environment into specific genetic programs, a property that renders them good candidates to be mediators of rapid adaptation changes of a species. Lipid-based morphogens, endocrine hormones, fatty acids and xenobiotics might act through this class of transcription factors making them regulators able to fine-tune physiological processes. Here we review the basic concepts and current knowledge on the process whereby small molecules act through nuclear receptors and contribute to transgenerational changes. Several molecules shown to cause transgenerational changes like phthalates, BPA, nicotine, tributylin bind and activate nuclear receptors like ERs, androgen receptors, glucocorticoid receptors or PPARγ. A specific subset of observations involving nuclear receptors has focused on the effects of environmental stress or maternal behaviour on the development of transgenerational traits. While these effects do not involve environmental ligands, they change the expression levels of Estrogen and glucocorticoid receptors of the second generation and consequently initiate an altered genetic program in the second generation. In this review we summarize the available literature about the role of nuclear receptors in transgenerational inheritance.

PRMT1 and PRMT8 regulate retinoic acid-dependent neuronal differentiation with implications to neuropathology.

Retinoids are morphogens and have been implicated in cell fate commitment of embryonic stem cells (ESCs) to neurons. Their effects are mediated by RAR and RXR nuclear receptors. However, transcriptional cofactors required for cell and gene-specific retinoid signaling are not known. Here we show that protein arginine methyl transferase (PRMT) 1 and 8 have key roles in determining retinoid regulated gene expression and cellular specification in a multistage neuronal differentiation model of murine ESCs. PRMT1 acts as a selective modulator, providing the cells with a mechanism to reduce the potency of retinoid signals on regulatory "hotspots." PRMT8 is a retinoid receptor target gene itself and acts as a cell type specific transcriptional coactivator of retinoid signaling at later stages of differentiation. Lack of either of them leads to reduced nuclear arginine methylation, dysregulated neuronal gene expression, and altered neuronal activity. Importantly, depletion of PRMT8 results in altered expression of a distinct set of genes, including markers of gliomagenesis. PRMT8 is almost entirely absent in human glioblastoma tissues. We propose that PRMT1 and PRMT8 serve as a rheostat of retinoid signaling to determine neuronal cell specification in a context-dependent manner and might also be relevant in the development of human brain malignancy.

Mapping the genomic binding sites of the activated retinoid X receptor in murine bone marrow-derived macrophages using chromatin immunoprecipitation sequencing.

Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) is a powerful technique to map the genomic location of a given chromatin bound factor (i.e., transcription factors, cofactors) or epigenetic marks, such as histone modification. The procedure is based on cross-linking of proteins to DNA followed by the capture of the protein-DNA complexes by "ChIP-grade" antibodies. In this chapter we describe in detail the experimental method developed in our laboratory to investigate in vivo the DNA-binding characteristics of a key heterodimeric nuclear receptor, the retinoid X receptor (RXR) in murine bone marrow-derived macrophages.

Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples.

MicroRNAs are involved in the regulation of various pathophysiological processes such as immune regulation and cancer. Next-generation sequencing methods enable us to monitor their presence in various types of samples but we need flexible methods for validating datasets generated by high-throughput methods. Here we describe the detailed protocols to be used with our MiRNA Primer Design Tool assay design system. The presented methods allow the flexible design of the oligonucleotides needed for the RT-qPCR detection of any variant of small regulatory RNA molecules from virtually any species. This method can be used to measure miRNA levels from formalin-fixed, paraffin-embedded (FFPE) samples and various body fluids. As an example, we show the results of the hsa-miR-515-3p, hsa-miR-325, and hsa-miR-155 quantification using a specific UPL probe (Universal Probe Library) and a stem-loop RT-qPCR assay. The small nucleolar RNA RNU43 is used as endogenous control for normalization of the results. Urine from healthy pregnant women and FFPE samples from patients diagnosed with colorectal cancer and treated with antibody-based anti-EGFR monotherapy were used as samples.