Journal articles: 'MiRNA reference gene selection'

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Lyu, Shiheng, Ying Yu, Shirong Xu, Weiwei Cai, Guixin Chen, Jianjun Chen, Dongming Pan, and Wenqin She. "Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri." Genes 11, no. 1 (December 23, 2019): 17. http://dx.doi.org/10.3390/genes11010017.

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MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.

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Korma, Workneh, Adane Mihret, Azeb Tarekegn, Yunhee Chang, Dasom Hwang, Tesfaye Sisay Tessema, and Hyeyoung Lee. "Identification of Circulating miR-22-3p and miR-93-5p as Stable Endogenous Control in Tuberculosis Study." Diagnostics 10, no. 11 (October 23, 2020): 868. http://dx.doi.org/10.3390/diagnostics10110868.

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The diagnosis and prognosis of tuberculosis remains challenging and necessitates the development of a new test that can accurately diagnose and monitor treatment responses. In this regard, miRNA is becoming a potential diagnostic and prognostic biomarker which differentiates treatment respondents from non-respondents for various non-infectious and infectious diseases, including tuberculosis. The concentration of miRNAs varies based on cell type, disease, and site of infection, implicating that selection of an optimal reference gene is crucial, and determines the quantification of transcript level and biological interpretation of the data. Thus, the study evaluated the stability and expression level of five candidate miRNAs (let-7i-5p, let-7a-5p, miRNA-16-5p, miRNA-22-3p and miRNA-93-5p), including U6 Small Nuclear RNA (RNU6B) to normalize circulating miRNAs in the plasma of 68 participants (26 healthy controls, 23 latent, and 19 pulmonary tuberculosis infected) recruited from four health centers and three hospitals in Addis Ababa, Ethiopia. The expression levels of miRNAs isolated from plasma of culture confirmed newly diagnosed pulmonary tuberculosis patients were compared with latently infected and non-infected healthy controls. The qPCR data were analyzed using four independent statistical tools: Best Keeper, Genorm, Normfinder and comparative delta-Ct methods, and the data showed that miRNA-22-3p and miRNA-93-5p were suitable plasma reference miRNAs in a tuberculosis study.

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Buonpane, Christie, Guillermo Ares, Beshoy Benyamen, Carrie Yuan, and Catherine J. Hunter. "Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease." Physiological Genomics 51, no. 5 (May 1, 2019): 169–75. http://dx.doi.org/10.1152/physiolgenomics.00126.2018.

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Pediatric inflammatory bowel disease (IBD) accounts for 10–15% of IBD and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA, miR), small noncoding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance, and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, is currently lacking. We hypothesize that appropriate normalization requires unique reference genes for different tissue and disease types. Through the study of 28 pediatric intestinal samples, we sought to create a protocol for selection of suitable endogenous reference genes. Candidate reference genes (miR-16, 193a, 27a, 103a, 191) were analyzed by RT-quantitative (q)PCR. Criteria used for designation of suitable reference genes were as follows: 1) ubiquitous: present in all tissue samples with quantification cycle value 15–35; 2) uniform expression: no differential expression between control and disease samples ( P > 0.05); 3) stability: stability value <0.5 by NormFinder. Our results suggest the use of miR-27a/191 for Crohn’s disease small bowel, none of the five candidate genes for Crohn’s disease colon, and miR-16/27a for ulcerative colitis. Additionally, target miR-874 had differential expression when normalized with different reference genes. Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.

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Yin, Zhimin, Fuliang Xie, Krystyna Michalak, Baohong Zhang, and Ewa Zimnoch-Guzowska. "Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y." Molecular and Cellular Probes 55 (February 2021): 101691. http://dx.doi.org/10.1016/j.mcp.2020.101691.

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Ghanbari, Sogol, Adel Salimi, Saeid Rahmani, Nahid Nafissi, Ali Sharifi-Zarchi, and Seyed Javad Mowla. "miR-361-5p as a promising qRT-PCR internal control for tumor and normal breast tissues." PLOS ONE 16, no. 6 (June 8, 2021): e0253009. http://dx.doi.org/10.1371/journal.pone.0253009.

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Background One of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples. Methods A multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we extended the scoring system for more than two biological sub-groups. TCGA BRCA samples were analyzed based on two grouping criteria: Tumor & Normal samples and Tumor subtypes. The top 10 most stable miRNAs were further investigated by differential expression and survival analysis. Then, we examined the expression level of the top scored miRNA (hsa-miR-361-5p) along with two commonly used ICs hsa-miR-16-5p and U48 on 34 pairs of Primary breast tumor and their adjacent normal tissues using qRT-PCR. Results According to our multi-component scoring system, hsa-miR-361-5p had the highest stability score in both grouping criteria and hsa-miR-16-5p showed significantly lower scores. Based on our qRT-PCR assay, while U48 was the most abundant IC, hsa-miR-361-5p had lower standard deviation and also was the only IC capable of detecting a significant up-regulation of hsa-miR-21-5p in breast tumor tissue. Conclusions miRNA-Seq data is a great source to discover stable ICs. Our results demonstrated that hsa-miR-361-5p is a highly stable miRNA in tumor and non-tumor breast tissue and we recommend it as a suitable reference gene for miRNA expression studies in breast cancer. Additionally, although hsa-miR-16-5p is a commonly used IC, it’s not a suitable one for breast cancer studies.

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Sippl, Christoph, Louisa Schoeneberger, Fritz Teping, Walter Schulz-Schaeffer, Steffi Urbschat, Ralf Ketter, and Joachim Oertel. "Impact of MiRNA-181a2 on the Clinical Course of IDH1 Wild Type Glioblastoma." Processes 9, no. 5 (April 21, 2021): 728. http://dx.doi.org/10.3390/pr9050728.

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Background: Recently, miRNA-181a2 could be identified as a major regulator of IDH1 expression in fat tissue. The IDH1 gene, its mutation and expression have a major impact on overall survival in patients with glioblastoma. The presented study aimed to investigate the effect of miRNA-181a2 on IDH1 expression in glioblastoma and on the prognosis of patients suffering from, for example, a tumor. Methods: A total of 74 glioblastoma specimens were analyzed for the expression of miRNA-181a2, acquired as fold change, using qRT-PCR. IDH1 protein expression was estimated via mRNA quantification. Eight post mortal, non-glioma related brain tissue specimens served as the control group. The results were correlated with relevant demographic and clinical aspects of the cohort. A TCGA dataset was used as an independent reference. Results: MiRNA-181a2 was significantly downregulated in tumor samples compared to the control group (p < 0.001). In the glioblastoma cohort, 63/74 (85.1%) showed an IDH1 wild type, while 11/74 (14.9%) patients harbored an IDH 1 mutation. In patients with IDH1 wild type glioblastoma, low miRNA-181a2 expression correlated with a prolonged overall survival (p = 0.019), also verifiable in an independent TCGA dataset. This correlation could not be identified for patients with an IDH1 mutation. MiRNA-181a2 expression tended to correlate inversely with IDH1 protein expression (p = 0.06). Gross total resection of the tumor was an independent marker for a prolonged survival (p = 0.03). Conclusion: MiRNA-181a2 seems to be a promising prognostic marker of selective glioblastoma patients with IDH1 wild type characteristics. This effect may be mediated via direct regulation of IDH1 expression.

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Ocłoń, Ewa, and Anna Hrabia. "Selection of the Most Stable Endogenous Control Genes for Microrna Quantitation in Chicken Ovarian Follicles." Annals of Animal Science 20, no. 1 (January 1, 2020): 109–23. http://dx.doi.org/10.2478/aoas-2019-0070.

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AbstractMicroRNAs (miRNAs or miRs) belong to a class of small non-coding RNAs of 19 to 24 nucleotides long that act as negative gene regulators at the post-transcriptional level. Quantitative PCR (q-PCR) is a commonly used technique in the profiling of miRs, and identification of reliable endogenous controls is crucial for proper data normalisation. To date, no study has been performed on reference miRs for the normalisation of miR expression in chicken ovarian tissues. Therefore, the aim of the present study was to experimentally identify the most stable reference mirs for normalisation of miR q-PCR expression data in the chicken ovary. Relying on high-throughput sequencing, five putative reference miR (let-7a-3p, miR-140a-3p, miR-22-5p, miR-33-5p, miR-99a-3p) were identified and subsequently analysed in a total of 66 tissue samples. The stability of candidate endogenous controls validated by the most widely used algorithms, geNorm, NormFinder, and BestKeeper, showed that let-7a-3p, miR-140a-3p, and miR-22-5p are the most appropriate choice of reference genes. Application of different normalisation approaches to the relative quantitation of randomly chosen miR-1552-5p in chicken ovarian follicles indicated the impact of the selected reference genes on miR expression. Further, the results revealed a downregulation of miR-1552-5p. In summary, the three identified endogenous reference miRs are suitable for profiling the miR expression in ovarian tissues of laying hens. Our findings provide valuable information for future miR expression studies in the avian ovary.

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Kim, Jin-Hyun, Joo-Seok Park, Chae-Young Lee, Min-Gyun Jeong, Jiu Liang Xu, Yongsoo Choi, Ho-Won Jung, and Hong-Kyu Choi. "Dissecting seed pigmentation-associated genomic loci and genes by employing dual approaches of reference-based and k-mer-based GWAS with 438 Glycine accessions." PLOS ONE 15, no. 12 (December 1, 2020): e0243085. http://dx.doi.org/10.1371/journal.pone.0243085.

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The soybean is agro-economically the most important among all cultivated legume crops, and its seed color is considered one of the most attractive factors in the selection-by-breeders. Thus, genome-wide identification of genes and loci associated with seed colors is critical for the precision breeding of crop soybeans. To dissect seed pigmentation-associated genomic loci and genes, we employed dual approaches by combining reference-based genome-wide association study (rbGWAS) and k-mer-based reference-free GWAS (rfGWAS) with 438 Glycine accessions. The dual analytical strategy allowed us to identify four major genomic loci (designated as SP1-SP4 in this study) associated with the seed colors of soybeans. The k-mer analysis enabled us to find an important recombination event that occurred between subtilisin and I-cluster B in the soybean genome, which could describe a special structural feature of ii allele within the I locus (SP3). Importantly, mapping analyses of both mRNAs and small RNAs allowed us to reveal that the subtilisin-CHS1/CHS3 chimeric transcripts generate and act as an initiator towards ‘mirtron (i.e., intron-harboring miRNA precursor)’-triggered silencing of chalcone synthase (CHS) genes. Consequently, the results led us to propose a working model of ‘mirtron-triggered gene silencing (MTGS)’ to elucidate a long-standing puzzle in the genome-wide CHS gene silencing mechanism. In summary, our study reports four major genomic loci, lists of key genes and genome-wide variations that are associated with seed pigmentation in soybeans. In addition, we propose that the MTGS mechanism plays a crucial role in the genome-wide silencing of CHS genes, thereby suggesting a clue to currently predominant soybean cultivars with the yellow seed coat. Finally, this study will provide a broad insight into the interactions and correlations among seed color-associated genes and loci within the context of anthocyanin biosynthetic pathways.

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Klinge, Carolyn M., Belinda J. Petri, and Kellianne M. Piell. "Epitranscriptomic Reader HNRNPA2B1 Confers Endocrine Resistance to Breast Cancer Cells." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A807—A808. http://dx.doi.org/10.1210/jendso/bvab048.1643.

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Abstract Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant metastatic disease remain unresolved. HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), an RNA binding protein that functions as reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is upregualted in tamoxifen- and fulvestrant-resistant, estrogen receptor (ERα)+ LCC9 and LY2 cells derived from MCF-7 endocrine-sensitive luminal A breast cancer cells (1). The miRNA-seq transcriptome of MCF-7 cells transiently overexpressing HNRNPA2B1 (A2B1) identified gene ontology (GO) pathways including “cellular response to steroid hormone signaling and estradiol” and “positive regulation of protein ser/thr kinase activity”. Modest (~ 4.5-fold) stable HNRNPA2B1 overexpression in MCF-7 cells (MCF-7-A2B1) results in ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not due to loss or decrease of ERα; in fact, ERα was increased. Conversely, transient knockdown of HNRNPA2B1 in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant while reducing ERα. MCF-7-A2B1 cells showed increased migration, invasion, clonogenicity, soft agar colony size, and markers of epithelial-to-mesenchymal transition. Like LCC9 cells, MCF-7-A2B1 cells showed activation of AKT and MAPK and high androgen receptor (AR). Treatment of MCF-7-A2B1 cells with either PI3K inhibitor Wortmannin or MEK inhibitor PD98059 inhibited soft agar colony formation and reduced colony size. Knockdown of HNRNPA2B1 in MCF-7-A2B1 reduces clonogenicity, but had no effect on the clonogenicity of either LCC9 or LY2 cells. These data suggest a role for HNRNPA2B1 in promoting the initiation of acquired endocrine-resistance by activating ser/thr kinase growth factor signaling pathways. Selective inhibition of HNRNPA2B1 may be a target to prevent acquistion of endocrine therapy resistance, but not treat established metastatic disease. Reference: (1) Klinge CM, Piell KM, Tooley CS, Rouchka EC. HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells. Scientific reports 2019; 9:9430

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Milani, Gloria, Tobia Lana, Silvia Bresolin, Francesca Paderi, Chiara Frasson, Giuseppe Basso, and Geertruy Kronnie. "Microvesicles Transcripts As Hallmark and Vector From Leukemic Parental Cells." Blood 120, no. 21 (November 16, 2012): 1459. http://dx.doi.org/10.1182/blood.v120.21.1459.1459.

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Abstract Abstract 1459 Microvesicles (MVs) are nano-sized lipid bodies (100–1000 nm in diameter) that are released by cells, both in vitro and in vivo. MVs are secreted in the extracellular space and as carriers of proteins, mRNA and miRNA represent vectors from donor to target cells involved in intercellular communication. MVs modulate the functional state of receiving cells through fusion with the target cell. In leukemia MVs were suggested to modulate the hematopoietic niche. In this study, we investigated the transcriptome of MVs released from leukemic cell lines. In particular, we analyzed K562, a BCR-ABL positive human erythromyeloblastoid leukemia cell line, and we collected RNA both from cells and MVs released in culture. Since many different methods have been described for microvesicles isolation and description of MV populations are often ambiguous, an accurate protocol has been developed in order to select a defined MVs population. In detail, for MVs isolation cell culture medium was centrifuged at 2500g for 15 minutes. These centrifugations allowed to delete cells and bigger bodies. Then supernatant was filtered by means of a 1.2um filter, in order to keep only vesicles of defined physical measure and to eliminate residual bigger vesicles, such as apoptotic bodies (>1000nm). The filtration allowed an accurate selection of a well defined MVs population. The filtered medium was then centrifuged at 18000g for 1h at 4°C. MVs were resuspended in Trizol for RNA isolation. Also RNA of cells, from which MVs have been released, was extracted using the Trizol method, according to manufacturer instructions. Cell line RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by means of NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). To check MVs RNA integrity we determined the ratio of 5' amplicons to 3' amplicons of housekeeping transcripts by Real Time PCR. We screened the presence of several housekeeping transcripts in MVs and selected GAPDH as reference. The GAPDH 5':3' amplicon ratio should equal 1 to ensure MVs RNA quality. Afterwards, reverse transcription–polymerase chain reaction (RT-PCR) amplification was performed. cDNA was synthesized from 1mg of total RNA. We analyzed the gene expression profile of K562 cell line and MVs, released from these cells, using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). Gene expression data have been compared between cells and MVs. Ninety-one percent of probe sets showed a similar expression (fold-change lower than 1.5) between cell line and MVs. Thirteen percent of these probes were recognized as present call probe sets in both cell line and MVs. Analysis of probe sets using DAVID Functional Annotation Bioinformatics Microarray Analysis revealed a conservation of MVs gene transcripts involved in pathways of cell function such as RNA processing, protein translation, aminoacid metabolism and cell respiration. Remarkably, in MVs we observed a high presence of gene transcripts coding for protein belonging to the Chronic Myeloid Leukemia pathway that are expressed downstream of the BCR-ABL tyrosin kinase fusion protein. The maintenance of this pathway in MVs highlights the intrinsic peculiarity of BCR-ABL positive K562 cells apparently also conserved in MVs mRNA outfit representing a hallmark of the parental cell from which they have been released. Furthermore, 3.8% of the probe sets resulted to be up-regulated in MVs compare to the cell line (fold-change higher than 1.5). In MVs, we observed a higher expression of genes belonging to cell communication pathways, adhesion and migration processes, membrane and ionic channels signals. In conclusion, we isolated MVs released by K562 leukemic cells using an accurate selection of the MV population based on physical measures and for the first time a whole transcriptome gene expression analysis has been performed comparing K562 cells and MVs. Moreover, we identified an enrichment of transcripts coding for proteins involved in several essential pathways in the MVs supporting the hypothesis of a functional selection from the parental cell transcriptome and underlining the relevant role of MVs as vehicles of messages to target cells. Disclosures: No relevant conflicts of interest to declare.

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Hu, Zihua, and Andrew E. Bruno. "The Influence of 3′UTRs on MicroRNA Function Inferred from Human SNP Data." Comparative and Functional Genomics 2011 (2011): 1–9. http://dx.doi.org/10.1155/2011/910769.

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MicroRNAs (miRNAs) regulate gene expression posttranscriptionally. Although previous efforts have demonstrated the functional importance of target sites on miRNAs, little is known about the influence of the rest of 3′ untranslated regions (3′UTRs) of target genes on microRNA function. We conducted a genome-wide study and found that the entire 3′UTR sequences could also play important roles on miRNA function in addition to miRNA target sites. This was evidenced by the fact that human single nucleotide polymorphisms (SNPs) on both seed target region and the rest of 3′UTRs of miRNA target genes were under significantly stronger negative selection, when compared to non-miRNA target genes. We also discovered that the flanking nucleotides on both sides of miRNA target sites were subject to moderate strong selection. A local sequence region of ~67 nucleotides with symmetric structure is herein defined. Additionally, from gene expression analysis, we found that SNPs and miRNA target sites on target sequences may interactively affect gene expression.

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Tonge, D. P., and T. W. Gant. "Evidence based housekeeping gene selection for microRNA-sequencing (miRNA-seq) studies." Toxicology Research 2, no. 5 (2013): 328. http://dx.doi.org/10.1039/c3tx50034a.

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Ragni, De Luca, Marmotti, and de Girolamo. "miR-26a-5p is a Stable Reference Gene for miRNA Studies in Chondrocytes from Developing Human Cartilage." Cells 8, no. 6 (June 22, 2019): 631. http://dx.doi.org/10.3390/cells8060631.

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miRNAs are emerging as key regulators of complex biological systems in several developmental processes. qRT-PCR is a powerful tool to quantitatively assess the profiles and modulation of miRNA expression. In the emerging field of cartilage maturation studies, from precursor to hypertrophic chondrocytes, few data about miRNA regulation are available, and no consensus on the best reference gene (RG) has been reached. This is a crucial pitfall since reliable outcomes depend on proper data normalization. The aim of this work was to identify reliable and stable miRNA RGs, basing the analysis on available high throughput qRT-PCR miRNA data (from the NCBI Gene Expression Omnibus database, GSE49152) obtained from human embryonic cartilage tissues enriched in the precursor, differentiated, and hypertrophic chondrocytes. Four normalization approaches were used, and the stability was quantified by combining BestKeeper, delta-Ct, geNorm, and NormFinder statistical tools. An integrated approach allowed to identify miR-26a-5p as the most stable RG and miR-212-3p as the worst one. RNU44, used in original dataset analysis, performed as second best RG. Applications of different normalization strategies significantly impacted the profiles and modulation of miRNA expression. Herein presented results point out the crucial need of a consensus on data normalization studies aimed at dissecting miRNA role in human cartilage development, to avoid the postulation of unreliable biological conclusions.

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Wessels, Jocelyn M., Andrew K. Edwards, Candace Zettler, and Chandrakant Tayade. "Selection and Validation of Reference Genes for miRNA Expression Studies during Porcine Pregnancy." PLoS ONE 6, no. 12 (December 12, 2011): e28940. http://dx.doi.org/10.1371/journal.pone.0028940.

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Verstraeten, Bruno, Lien De Smet, Tina Kyndt, and Tim De Meyer. "Selection of miRNA reference genes for plant defence studies in rice (Oryza sativa)." Planta 250, no. 6 (October 3, 2019): 2101–10. http://dx.doi.org/10.1007/s00425-019-03289-x.

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Zhang, Yingting, Jinyu Xue, Lijuan Zhu, Hailiang Hu, Junjie Yang, Jiebing Cui, and Jin Xu. "Selection and Optimization of Reference Genes for MicroRNA Expression Normalization by qRT-PCR in Chinese Cedar (Cryptomeria fortunei) under Multiple Stresses." International Journal of Molecular Sciences 22, no. 14 (July 6, 2021): 7246. http://dx.doi.org/10.3390/ijms22147246.

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MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including Cryptomeria fortunei, has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (U6, 5S and 18S) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in C. fortunei under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and U6 were the most stable RGs for studying C. fortunei miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of C. fortunei to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species.

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Piestrzynska-Kajtoch, Agata, Grzegorz Smołucha, Maria Oczkowicz, Anna Kycko, Mirosław P. Polak, Wojciech Kozaczyński, Anna Kozubska-Sobocińska, Jan F. Żmudziński, and Barbara Rejduch. "The Reference Gene Selection to Study PRNP Gene Expression in Sheep." Folia Biologica 65, no. 3 (December 21, 2017): 164–70. http://dx.doi.org/10.3409/fb65_3.165.

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Larabi, Anaïs, Laurène Salesse, Charlotte Cordonnier, Lucie Etienne-Mesmin, Nicolas Barnich, Guillaume Dalmasso, and Hang Thi Thu Nguyen. "Differential miRNA-Gene Expression in M Cells in Response to Crohn’s Disease-Associated AIEC." Microorganisms 8, no. 8 (August 7, 2020): 1205. http://dx.doi.org/10.3390/microorganisms8081205.

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Adherent-invasive Escherichia coli (AIEC), which abnormally colonize the ileal mucosa of Crohn’s disease (CD) patients, are able to invade intestinal epithelial cells (IECs) and translocate through M cells overlying Peyer’s patches. The levels of microRNA (miRNA) and gene expression in IECs and M cells upon AIEC infection have not been investigated. Here, we used human intestinal epithelial Caco-2 monolayers and an in vitro M-cell model of AIEC translocation to analyze comprehensive miRNA and gene profiling under basal condition and upon infection with the reference AIEC LF82 strain. Our results showed that AIEC LF82 translocated through M cells but not Caco-2 monolayers. Both differential gene expression and miRNA profile in M cells compared to Caco-2 cells were obtained. In addition, AIEC infection induces changes in gene and miRNA profiles in both Caco-2 and M cells. In silico analysis showed that certain genes dysregulated upon AIEC infection were potential targets of AIEC-dysregulated miRNAs, suggesting a miRNA-mediated regulation of gene expression during AIEC infection in Caco-2, as well as M cells. This study facilitates the discovery of M cell-specific and AIEC response-specific gene-miRNA signature and enhances the molecular understanding of M cell biology under basal condition and in response to infection with CD-associated AIEC.

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Krishna Himmatbhai Goyani, Shalin Vaniawala, and Pratap Narayan Mukhopadhyaya. "Mutations within the Open Reading Frame (ORF) including Ochre stop codon of the Surface Glycoprotein gene of SARS-CoV-2 virus erase potential seed location motifs of human non-coding microRNAs." World Journal of Advanced Research and Reviews 10, no. 3 (June 30, 2021): 370–91. http://dx.doi.org/10.30574/wjarr.2021.10.3.0288.

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MicroRNA are short and non-coding RNA, 18-25 nucleotides in length. They are produced at the early stage of viral infection. The roles played by cellular miRNAs and miRNA-mediated gene-silencing in the COVID-19 epidemic period is critical in order to develop novel therapeutics. We analyzed SARS-CoV-2 Surface Glycoprotein (S) nucleotide sequence originating from India as well as Iran, Australia, Germany, Italy, Russia, China, Japan and Turkey and identified mutation in potential seed location of several human miRNA. Seventy single nucleotide polymorphisms (SNP) were detected in the S gene out of which, 36, 32 and 2 were cases of transitions, transversions and deletions respectively. Eleven human miRNA targets were identified on the reference S gene sequence with a score >80 in the miRDB database. Mutation A845S erased a common binding site of 7 human miRNA (miR-195-5p, miR-16-5p, miR-15b-5p, miR-15a-5p, miR-497-5p, miR-424-5p and miR-6838-5p). A synonymous mutation altered the wild type Ochre stop codon within the S gene sequence (Italy) to Opal thereby changing the seed sequence of miR-511-3p. Similar (synonymous) mutations were detected at amino acid position 659 and 1116 of the S gene where amino acids serine and threonine were retained, abolishing potential seed location for miR-219a-1-3p and miR-20b-3p respectively. The significance of this finding in reference to the strategy to use synthetic miRNA combinations as a novel therapeutic tool is discussed.

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Desvignes, Thomas, Jason Sydes, Jerôme Montfort, Julien Bobe, and John H. Postlethwait. "Evolution after Whole-Genome Duplication: Teleost MicroRNAs." Molecular Biology and Evolution 38, no. 8 (April 19, 2021): 3308–31. http://dx.doi.org/10.1093/molbev/msab105.

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Abstract MicroRNAs (miRNAs) are important gene expression regulators implicated in many biological processes, but we lack a global understanding of how miRNA genes evolve and contribute to developmental canalization and phenotypic diversification. Whole-genome duplication events likely provide a substrate for species divergence and phenotypic change by increasing gene numbers and relaxing evolutionary pressures. To understand the consequences of genome duplication on miRNA evolution, we studied miRNA genes following the teleost genome duplication (TGD). Analysis of miRNA genes in four teleosts and in spotted gar, whose lineage diverged before the TGD, revealed that miRNA genes were retained in ohnologous pairs more frequently than protein-coding genes, and that gene losses occurred rapidly after the TGD. Genomic context influenced retention rates, with clustered miRNA genes retained more often than nonclustered miRNA genes and intergenic miRNA genes retained more frequently than intragenic miRNA genes, which often shared the evolutionary fate of their protein-coding host. Expression analyses revealed both conserved and divergent expression patterns across species in line with miRNA functions in phenotypic canalization and diversification, respectively. Finally, major strands of miRNA genes experienced stronger purifying selection, especially in their seeds and 3′-complementary regions, compared with minor strands, which nonetheless also displayed evolutionary features compatible with constrained function. This study provides the first genome-wide, multispecies analysis of the mechanisms influencing metazoan miRNA evolution after whole-genome duplication.

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Li, Xiao-Shuang, Hong-Lan Yang, Dao-Yuan Zhang, Yuan-Ming Zhang, and Andrew J. Wood. "Reference Gene Selection in the Desert Plant Eremosparton songoricum." International Journal of Molecular Sciences 13, no. 6 (June 7, 2012): 6944–63. http://dx.doi.org/10.3390/ijms13066944.

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Santosh P., Shinde, Neelima Arora, Pranjal Sarma, Manika Pal-Bhadra, and Utpal Bhadra. "Interaction Map and Selection of microRNA Targets in Parkinson's Disease-Related Genes." Journal of Biomedicine and Biotechnology 2009 (2009): 1–11. http://dx.doi.org/10.1155/2009/363145.

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Parkinson's disease (PD) is a complex multigenic neurodisorder frequently occurring in elderly persons. To investigate noncoding tiny microRNA mediated gene regulation, miRanda (version 1.0b) was used to predict human miRNA target sites on selected 29 genes related to PD. To verify output generated from miRanda, a similar analysis was performed only for microRNA target sites in3′UTR using TargetScan (version 5.1). Data extracted by miRanda elucidates the mode of microRNA action based on the location of target sites in the Parkinson genes. Sites prone to action of multiple miRNAs were identified as “hot spots.” Important properties of each miRNA including multiplicity and cooperativity appear to contribute towards a complex interplay between miRNAs and their targets. Two sets of predicted results were explored for the occurrence of target sites of 112 miRNAs expressed in midbrain. Overall, convergence of results predicted by two algorithms revealed that 48 target sites for midbrain-specific miRNA occur in close proximity in 9 genes. This study will pave a way for selection of potential miRNA candidates for Parkinson's disease-related genes for quick therapeutic applications and diagnosis.

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Schwarzenbach, Heidi, Andreia Machado da Silva, George Calin, and Klaus Pantel. "Data Normalization Strategies for MicroRNA Quantification." Clinical Chemistry 61, no. 11 (November 1, 2015): 1333–42. http://dx.doi.org/10.1373/clinchem.2015.239459.

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Abstract BACKGROUND Different technologies, such as quantitative real-time PCR or microarrays, have been developed to measure microRNA (miRNA) expression levels. Quantification of miRNA transcripts implicates data normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data. CONTENT In this review article we discuss the reliability of the use of small RNAs, commonly reported in the literature as miRNA expression normalizers, and compare different strategies used for data normalization. SUMMARY A workflow strategy is proposed for normalization of miRNA expression data in an attempt to provide a basis for the establishment of a global standard procedure that will allow comparison across studies.

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Matsuyama, Hironori, and Hiroshi I. Suzuki. "Systems and Synthetic microRNA Biology: From Biogenesis to Disease Pathogenesis." International Journal of Molecular Sciences 21, no. 1 (December 24, 2019): 132. http://dx.doi.org/10.3390/ijms21010132.

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MicroRNAs (miRNAs) are approximately 22-nucleotide-long, small non-coding RNAs that post-transcriptionally regulate gene expression. The biogenesis of miRNAs involves multiple steps, including the transcription of primary miRNAs (pri-miRNAs), nuclear Drosha-mediated processing, cytoplasmic Dicer-mediated processing, and loading onto Argonaute (Ago) proteins. Further, miRNAs control diverse biological and pathological processes via the silencing of target mRNAs. This review summarizes recent findings regarding the quantitative aspects of miRNA homeostasis, including Drosha-mediated pri-miRNA processing, Ago-mediated asymmetric miRNA strand selection, and modifications of miRNA pathway components, as well as the roles of RNA modifications (epitranscriptomics), epigenetics, transcription factor circuits, and super-enhancers in miRNA regulation. These recent advances have facilitated a system-level understanding of miRNA networks, as well as the improvement of RNAi performance for both gene-specific targeting and genome-wide screening. The comprehensive understanding and modeling of miRNA biogenesis and function have been applied to the design of synthetic gene circuits. In addition, the relationships between miRNA genes and super-enhancers provide the molecular basis for the highly biased cell type-specific expression patterns of miRNAs and the evolution of miRNA–target connections, while highlighting the importance of alterations of super-enhancer-associated miRNAs in a variety of human diseases.

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Scholtz, Jakobus J., and Botma Visser. "Reference gene selection for qPCR gene expression analysis of rust-infected wheat." Physiological and Molecular Plant Pathology 81 (January 2013): 22–25. http://dx.doi.org/10.1016/j.pmpp.2012.10.006.

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Jia, Wanjun, Yabin Zhang, and Ruian Wang. "miRNA-206 Regulates EVI1 Gene Expression, Akt Signaling Pathway and Cell Biological Behavior in Gastric Cancer Cells." Journal of Biomaterials and Tissue Engineering 9, no. 10 (December 1, 2019): 1381–87. http://dx.doi.org/10.1166/jbt.2019.2163.

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To investigate the impact of miRNA-206 on the transcriptional expression of EVI1 gene and activation of Akt/JNK signaling pathway in gastric cancer cells, and to provide a new idea for gene-targeted therapy of gastric cancer. The miRNA-206 transfection experiment was firstly used to verify the regulation of EVI1. The experiment was divided into three groups: miRNA-206 mimics (100 nM), miRNA-206 inhibitor (100 nM), miR-NC (100 nM), and transfected into gastric cancer cells sgc7901, Western blot. EVI1 protein expression was detected; then the signal transduction and biological behavior of the cells were verified by miRNA-206 lentiviral vector transfection experiments. The experiment was divided into three groups: pLB-miRNA-206 group, empty vector group and control group (sgc7901 cell group). miRNA-206 and EVI1 mRNA levels were detected by real-time fluorescence quantitative (RT-PCR), and p-Akt and p-JNK were detected by Western blot. Protein expression, cell proliferation was quantified by MTT assay, and the alteration of cell cycle were detected by flow cytometry. miRNA-206 may affect the cell proliferation and division cycle by targeting the regulation of EVI1 transcriptional gene expression and activation of Akt/JNK signaling pathway in gastric cancer cells, and it is expected to provide an important selection site for gene-targeted therapy of gastric cancer.

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Johansen, Ilona, and Rune Andreassen. "Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar)." BMC Research Notes 8, no. 1 (2014): 945. http://dx.doi.org/10.1186/1756-0500-7-945.

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Mahdipour, M., H. T. A. Van Tol, T. A. E. Stout, and B. A. J. Roelen. "225 VALIDATION OF REFERENCE microRNAs FOR NORMALIZING EXPRESSION DATA GENERATED BY QUANTITATIVE PCR." Reproduction, Fertility and Development 27, no. 1 (2015): 202. http://dx.doi.org/10.1071/rdv27n1ab225.

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Small noncoding RNAs such as microRNAs (miRNA) act as posttranscriptional regulators of numerous gene targets. The level of expression of these epigenetic factors can regulate the stability and translation of target mRNA molecules. Quantification of miRNA expression levels can therefore help us understand biological processes such as those regulating oocyte and pre-implantation embryo development. To date, most studies that have examined miRNA expression levels have used expression of either reference mRNAs or U6 spliceosomal RNA or miRNA Let-7a for normalization. We analysed the suitability of several miRNAs as potential expression normalizers in bovine oocytes and early embryos. Bovine oocytes at the germinal vesicle and metaphase II stages, and zygotes, 2-cell, 4-cell and 8-cell embryos, morulae, and blastocysts were collected during three in vitro fertilization replicates. qPCR was performed to quantify expression of miR-93, miR-103a, miR-26a, miR-191, miR-23b, Let-7a, and U6. The average starting material for each sample was determined with the help of specific standard curves made for each primer set. Subsequently, geNorm software was used to identify a set of stably transcribed miRNAs. Surprisingly, U6 spliceosomal RNA and Let-7a, which are widely used to normalise miRNA expression levels, were not stably expressed and were therefore considered unsuitable for normalization. Stepwise removal to determine the optimal number of reference miRNAs identified miR-93 and miR-103a as the most stably expressed. It is concluded that the combination of miR-93 and miR-103a can be used for normalizing miRNA expression for qPCR experiments on bovine oocytes and pre-implantation embryos.

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Zárybnický, Tomáš, Petra Matoušková, Martin Ambrož, Zdeněk Šubrt, Lenka Skálová, and Iva Boušová. "The Selection and Validation of Reference Genes for mRNA and microRNA Expression Studies in Human Liver Slices Using RT-qPCR." Genes 10, no. 10 (September 28, 2019): 763. http://dx.doi.org/10.3390/genes10100763.

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The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with β-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and β-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

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Tang, Xiaoli, Hongyan Wang, Chuyang Shao, and Hongbo Shao. "Reference Gene Selection for qPCR Normalization ofKosteletzkya virginicaunder Salt Stress." BioMed Research International 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/823806.

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Kosteletzkya virginica(L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene inK. virginicawhich showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA),β-actin (ACT),α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and18SrRNAwas assessed to be the most stable reference gene in this study. However,TUAwas identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies inK. virginica.

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Fragoulis, Athanassios, Kristina Biller, Stephanie Fragoulis, Dennis Lex, Stefan Uhlig, and Lucy Kathleen Reiss. "Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury." International Journal of Molecular Sciences 22, no. 15 (July 22, 2021): 7853. http://dx.doi.org/10.3390/ijms22157853.

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qRT-PCR still remains the most widely used method for quantifying gene expression levels, although newer technologies such as next generation sequencing are becoming increasingly popular. A critical, yet often underappreciated, problem when analysing qRT-PCR data is the selection of suitable reference genes. This problem is compounded in situations where up to 25% of all genes may change (e.g., due to leukocyte invasion), as is typically the case in ARDS. Here, we examined 11 widely used reference genes for their suitability in commonly used models of acute lung injury (ALI): ventilator-induced lung injury (VILI), in vivo and ex vivo, lipopolysaccharide plus mechanical ventilation (MV), and hydrochloric acid plus MV. The stability of reference gene expression was determined using the NormFinder, BestKeeper, and geNorm algorithms. We then proceeded with the geNorm results because this is the only algorithm that provides the number of reference genes required to achieve normalisation. We chose interleukin-6 (Il-6) and C-X-C motif ligand 1 (Cxcl-1) as the genes of interest to analyse and demonstrate the impact of inappropriate normalisation. Reference gene stability differed between the ALI models and even within the subgroup of VILI models, no common reference gene index (RGI) could be determined. NormFinder, BestKeeper, and geNorm produced slightly different, but comparable results. Inappropriate normalisation of Il-6 and Cxcl1 gene expression resulted in significant misinterpretation in all four ALI settings. In conclusion, choosing an inappropriate normalisation strategy can introduce different kinds of bias such as gain or loss as well as under- or overestimation of effects, affecting the interpretation of gene expression data.

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Noh, Yunjeong, Woncheoul Park, Han-Ha Chai, and Dajeong Lim. "Reference gene selection for gene expression study in tissues of long-tailed chickens." Journal of Biomedical and Translational Research 21, no. 4 (December 2020): 152–64. http://dx.doi.org/10.12729/jbtr.2020.21.4.152.

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Lallemant, Benjamin, Alexandre Evrard, Christophe Combescure, Heliette Chapuis, Guillaume Chambon, Caroline Raynal, Christophe Reynaud, et al. "Reference gene selection for head and neck squamous cell carcinoma gene expression studies." BMC Molecular Biology 10, no. 1 (2009): 78. http://dx.doi.org/10.1186/1471-2199-10-78.

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Weickert, Cynthia Shannon, Donna Sheedy, Debora A. Rothmond, Irina Dedova, Samantha Fung, Therese Garrick, Jenny Wong, et al. "Selection of Reference Gene Expression in a Schizophrenia Brain Cohort." Australian & New Zealand Journal of Psychiatry 44, no. 1 (January 2010): 59–70. http://dx.doi.org/10.3109/00048670903393662.

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Vorachek, William R., Gerd Bobe, and Jean A. Hall. "Reference gene selection for quantitative PCR studies in bovine neutrophils." Advances in Bioscience and Biotechnology 04, no. 10 (2013): 6–14. http://dx.doi.org/10.4236/abb.2013.410a3002.

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Rothmond, Debora A., Samantha J. Fung, Jenny Wong, Carlotta Duncan, Shan-Yuan Tsai, and Cynthia S. Weickert. "SELECTION OF REFERENCE GENE EXPRESSION IN A SCHIZOPHRENIA BRAIN COHORT." Schizophrenia Research 117, no. 2-3 (April 2010): 372. http://dx.doi.org/10.1016/j.schres.2010.02.663.

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Jin, Xuehua, Jianxin Fu, Silan Dai, Yi Sun, and Yan Hong. "Reference gene selection for qPCR analysis in cineraria developing flowers." Scientia Horticulturae 153 (April 2013): 64–70. http://dx.doi.org/10.1016/j.scienta.2013.01.023.

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Al-Sabah, A., P. Stadnik, S. J. Gilbert, V. C. Duance, and E. J. Blain. "Importance of reference gene selection for articular cartilage mechanobiology studies." Osteoarthritis and Cartilage 24, no. 4 (April 2016): 719–30. http://dx.doi.org/10.1016/j.joca.2015.11.007.

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Vorachek, William, Hugejiletu, Gerd Bobe, and Jean Hall. "Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils." International Journal of Molecular Sciences 14, no. 6 (May 30, 2013): 11484–95. http://dx.doi.org/10.3390/ijms140611484.

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Aggarwal, Anu, Manu Jamwal, Ganesh K. Viswanathan, Prashant Sharma, ManUpdesh S. Sachdeva, Deepak Bansal, Pankaj Malhotra, and Reena Das. "Optimal Reference Gene Selection for Expression Studies in Human Reticulocytes." Journal of Molecular Diagnostics 20, no. 3 (May 2018): 326–33. http://dx.doi.org/10.1016/j.jmoldx.2018.01.009.

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Martínez-Beamonte, Roberto, María A. Navarro, Ana Larraga, Mark Strunk, Cristina Barranquero, Sergio Acín, Mario A. Guzman, Pablo Iñigo, and Jesús Osada. "Selection of reference genes for gene expression studies in rats." Journal of Biotechnology 151, no. 4 (February 2011): 325–34. http://dx.doi.org/10.1016/j.jbiotec.2010.12.017.

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Radonić, Aleksandar, Stefanie Thulke, Ian M. Mackay, Olfert Landt, Wolfgang Siegert, and Andreas Nitsche. "Guideline to reference gene selection for quantitative real-time PCR." Biochemical and Biophysical Research Communications 313, no. 4 (January 2004): 856–62. http://dx.doi.org/10.1016/j.bbrc.2003.11.177.

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Gresner, Sylwia M., Ewa Golanska, Dominika Kulczycka-Wojdala, Dariusz J. Jaskolski, Wielislaw Papierz, and Pawel P. Liberski. "Selection of reference genes for gene expression studies in astrocytomas." Analytical Biochemistry 408, no. 1 (January 2011): 163–65. http://dx.doi.org/10.1016/j.ab.2010.09.010.

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Ding, Yan, Hengyi Sun, Ran Zhang, Qin Yang, Yuantao Liu, Xiaonan Zang, and Xuecheng Zhang. "Selection of reference gene from Gracilaria lemaneiformis under temperature stress." Journal of Applied Phycology 27, no. 3 (October 31, 2014): 1365–72. http://dx.doi.org/10.1007/s10811-014-0423-2.

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Kern, Fabian, Ernesto Aparicio-Puerta, Yongping Li, Tobias Fehlmann, Tim Kehl, Viktoria Wagner, Kamalika Ray, et al. "miRTargetLink 2.0—interactive miRNA target gene and target pathway networks." Nucleic Acids Research 49, W1 (May 1, 2021): W409—W416. http://dx.doi.org/10.1093/nar/gkab297.

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Abstract Which genes, gene sets or pathways are regulated by certain miRNAs? Which miRNAs regulate a particular target gene or target pathway in a certain physiological context? Answering such common research questions can be time consuming and labor intensive. Especially for researchers without computational experience, the integration of different data sources, selection of the right parameters and concise visualization can be demanding. A comprehensive analysis should be central to present adequate answers to complex biological questions. With miRTargetLink 2.0, we develop an all-in-one solution for human, mouse and rat miRNA networks. Users input in the unidirectional search mode either a single gene, gene set or gene pathway, alternatively a single miRNA, a set of miRNAs or an miRNA pathway. Moreover, genes and miRNAs can jointly be provided to the tool in the bidirectional search mode. For the selected entities, interaction graphs are generated from different data sources and dynamically presented. Connected application programming interfaces (APIs) to the tailored enrichment tools miEAA and GeneTrail facilitate downstream analysis of pathways and context-annotated categories of network nodes. MiRTargetLink 2.0 is freely accessible at https://www.ccb.uni-saarland.de/mirtargetlink2.

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Bhaumik, Panchalee, Chandrasekhar Gopalakrishnan, Balu Kamaraj, and Rituraj Purohit. "Single Nucleotide Polymorphisms in MicroRNA Binding Sites: Implications in Colorectal Cancer." Scientific World Journal 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/547154.

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Cancer is a complex genetic disorder, characterised by uncontrolled cell proliferation and caused by altered expression of oncogenes and tumour suppressor genes. When cell proliferation pertains to colon, it is called colorectal cancer. Most of colorectal cancer causing genes are potential targets for the miRNA (microRNA) that bind to 3′UTR (untranslated regions) of mRNA and inhibit translation. Mutations occurring in miRNA binding regions can alter the miRNA, mRNA combination, and can alter gene expression drastically. We hypothesized that 3′UTR mutation in miRNA binding site could alter the miRNA, mRNA interaction, thereby altering gene expression. Altered gene expression activity could promote tumorigenesis in colon. Therefore, we formulated a systematic in silico procedure that integrates data from various databases, followed rigorous selection criteria, and identified mutations that might alter the expression levels of cancer causing genes. Further we performed expression analysis to shed light on the potential tissues that might be affected by mutation, enrichment analysis to find the metabolic functions of the gene, and network analysis to highlight the important interactions of cancer causing genes with other genes to provide insight that complex network will be disturbed upon mutation. We provide in silico evidence for the effect of these mutations in colorectal cancer.

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Abraham, Shaji, Raul Teruel Montoya, Leonard C. Edelstein, Steven E. McKenzie, and Paul F. Bray. "Identification of Reference Genes for miRNA Profiling in Hematopoietic Cell Lineages." Blood 120, no. 21 (November 16, 2012): 3330. http://dx.doi.org/10.1182/blood.v120.21.3330.3330.

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Abstract Abstract 3330 MicroRNAs (miRNAs) are important regulators of gene expression involved in virtually all physiological and pathological processes. Transcriptional profiling with subsequent bioinformatic analysis is increasingly used to identify miRNAs critical for hematopoiesis, normal blood cell function and hematological diseases. The biological variation observed in differential expression of these RNAs has been useful for better understanding of disease mechanism, and for identifying potential biomarkers and therapeutic targets. However, conclusions about the significance of measured biological variation is only legitimate when the RNA expression levels are normalized to internal controls, and most miRNA profiling data analyses are based on assumptions that have not been validated. For example, the expression level of U6 snRNA is often used to normalize expression across comparison tissues. However, it is not known whether U6 snRNA is expressed at equivalent levels among these cells or sample groups, and no reliable reference genes have been identified in cells from different hematopoietic lineages. The goal of this study was to identify reference miRNAs with the least variation for peripheral blood T-cells, B-cells, granulocytes and platelets. Subsets of cells were obtained from the peripheral blood of 5 healthy donors using density centrifugation followed by immunoselection. High purity (>97%) of the cells was verified by flow cytometry. Total RNA was extracted with Trizol and miRNA profiled by Nanostring technology (Nanostring Technologies, Denver, CO). Two different statistical algorithms - the NormFinder (NF) and Coefficient of variation (CV) methods - were used to identify miRNA reference genes with high stability within and among cell types. Geometric mean normalized raw data was analyzed by NF and CV, and candidate normalizer genes were selected based on three criteria: (1) stability measure of the variation in miRNA values (low values being more stable) (2) present in the top 10 candidate normalizer genes by both the NF and CV methods, and (3) a moderate expression value of 300–1000 counts (range = 50–12,000 counts for 95% of miRNAs). The last criterion is important because variation in a very low abundance reference gene would have an inappropriately large effect on normalization of the data. The reference miRNAs identified were hsa-miR-30b and hsa-miR-151-5p for B-cells, hsa-miR-484 and hsa-miR-425 for platelets, hsa-miR-301a, hsa-miR-30d and hsa-miR-424 for granulocytes, and hsa-miR-140-3p and hsa-miR-101 for T-cells. Hsa-let-7b and hsa-miR-423-3p were identified as common normalizers across T-cells, B-cells, platelets and granulocytes with stability values of 0.207 and 0.331 by NormFinder and 0.0599 and 0.0591 by CV method as shown in Fig. 1 and Fig. 2. Both hsa-let-7b and hsa-miR-423-3p were validated by RT-PCR to be stable normalizers (CV of Ct values were 9% and 12%, respectively) across T-cells, B-cells, platelets and granulocytes. Notably, hsa-let-7b showed lower variation across cell types than U6 snRNA, indicating hsa-let-7b is a more reliable reference gene for quantification of miRNA data from hematopoietic cells. In summary, we used a rigorous and formal approach to identify miRNAs in different hematopoietic lineages that can be used as appropriate reference transcripts for genome-wide profiling studies. Most ideally, this approach can be used for any new transcriptome data set. In addition, we provide ideal reference miRNAs for comparisons within or among different hematopoietic lineages. Normalization with the reference genes identified in this report, will allow more reliable and accurate determination of the biological variation involving differential expression of hematopoietic miRNAs than currently used normalizers. Disclosures: No relevant conflicts of interest to declare.

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Wang, Xiaodong, and Jun Tian. "A Gene Selection Method for Cancer Classification." Computational and Mathematical Methods in Medicine 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/586246.

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This paper proposes a method to select a set of genes from a large number of genes with the ability of classifying types of diseases. The proposed gene selection method is designed according to correlation analysis and the concept of 95% reference range. The method is very simple and uses the information of all genes. We have used the method in leukemia patients and achieved good classification results.

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Pushkin, Anton, Oleg I. Kit, Eduard Evgenevich Rostorguev, David H. Porksheyan, Natalya S. Kuznetsova, and Sergey E. Kavitskiy. "Aberrant miRNA expression in glioma." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e13506-e13506. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13506.

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e13506 Background: Gliomas are the most common type of primary brain tumors, with a high level of recurrence and mortality. The purpose of the study was to determine the expression profile of micro-RNA targeting components of the Hedgehog, Notch, Wnt, EGFR, TGFβ, HIF1α signaling pathways according to MirTarBase, miRDB, TargetScan. Methods: The study included 30 patients (15 women and 15 men) aged 27 to 76 years with histologically verified glial brain tumors. The RT2-qPCR method in operational biopsy specimens determined the relative expression of 12 micro-RNAs: hsa-miR-17-3p, hsa-miR-20a-3p, hsa-miR-326, hsa-miR-330-3p, hsa-miR- 107, hsa-miR-143-3p, hsa-let-7a-5p, hsa-miR-146a-5p, hsa-miR-29a-3p, hsa-miR-92a-1-5p, hsa-miR-26b-3p, hsa-miR-96-5p. Hsa-miR-7-5p and hsa-miR-126-3p were used as reference micro-RNA. Results: A statistically significant increase in the expression of hsa-miR-143-3p, hsa-miR-146a-5p and hsa-miR-92a-1-5p by 2, 2.2 and 2.9 times, respectively, was found, as well as a decrease in the expression of hsa-miR-330- 3p by 4.1 times in tumor tissue of the brain relative to normal tissue (p < 0.05). Reduced expression of micro-RNA hsa-miR-330-3p may increase the activity of the VEGFA gene, which leads to intensive vascularization of the tumor. In addition, hsa-miR-330-3p is a validated negative regulator of the E2F1 gene known as a regulator of the cell cycle and tumor suppressor proteins. The TRAF6 gene is the direct validated target of hsa-miR-146a-5p, and its overexpression is associated with the patient's chemoresistance to temozolomide. Increased hsa-miR-143-3p micro-RNA expression can reduce the activity of the EGLN1 gene that also mediates the adaptation of tumor cells to hypoxic conditions. Hsa-miR-92a-1-5p micro-RNA is a negative regulator of PTEN gene expression. Conclusions: A significant decrease in hsa-miR-330-3p expression and an increase in hsa-miR-146a-5p, hsa-miR-143-3p, and hsa-miR-92a-1-5p, which was found in gliomas, can potentially be used to develop prognostic markers and therapeutic targets for targeted therapy.

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Wu, Qian, Zuhong Lu, Hailing Li, Jiafeng Lu, Li Guo, and Qinyu Ge. "Next-Generation Sequencing of MicroRNAs for Breast Cancer Detection." Journal of Biomedicine and Biotechnology 2011 (2011): 1–7. http://dx.doi.org/10.1155/2011/597145.

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It is reported that different microRNA (miRNA) profiles can be detected in the blood of cancer patients. We investigated that whether the key serum miRNAs could discriminate patients with and without breast cancer. This study was divided into three parts: (1) miRNA marker discovery using SOLiD sequencing-based miRNA profiling on cancerous and adjacent noncancerous breast tissue of one breast cancer patient; (2) marker selection and validation by real-time PCR on a small set of serum; (3) gene ontology analysis of the key miRNA target genes. Of genome-wide tissue miRNA expression analysis, five miRNAs were found to be altered more than fivefold by SOLiD sequencing (i.e., miR-29a, miR-23a, miR-23b, miR-192, and miR-21). All the five miRNAs were validated on the 20 breast cancer patients and 20 controls. miR-29a and miR-21 were significantly increased in the serum of breast cancer patients (). Gene ontology analysis of the target genes revealed enrichment for special biological process categories, that is, signal transduction, development, apoptosis, cell proliferation, and cell adhesion. SOLiD sequencing provides a promising method for cancer-related miRNA profiling. Serum miRNAs may be useful biomarkers for breast cancer detection.

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Journal articles on the topic 'MiRNA reference gene selection'

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