Journal: PLoS ONE

Article Title: Microarray profiling of circular RNAs in human papillary thyroid carcinoma

doi: 10.1371/journal.pone.0170287

Figure Lengend Snippet: (A) The targeted pathway heatmap analysis revealed significant correlations (depicted in red) between cancer-related pathways and two miRNA clusters: the miR-141-3p/miR-15a-5p/miR-23b-3p cluster and the miR-200a-3p/miR-214-3p cluster. The color-coded legend depicts the associated log-scaled p -values. (B) Based on the Arraystar-derived network map of circRNA-miRNA interactions, the candidate circRNA hsa_circRNA_100395 was predicted to interact with the two cancer-related miRNAs: hsa-miR-141-3p and hsa-miR-200a-3p. (C) Seed sequence matching predicts the direct interaction of hsa_circRNA_100395 with miR-141-3p and miR-200a-3p.

Article Snippet: Therefore, Arraystar miRNA target prediction software (Arraystar), which is based on TargetScan and miRanda algorithms [ ], was applied to predict putative circRNA/miRNA interactions for the 12 differential circRNAs identified from the microarray and qRT-PCR validation experiments.

Techniques: Derivative Assay, Sequencing

Journal: Frontiers in Oncology

Article Title: TRPM7 Induces Mechanistic Target of Rap1b Through the Downregulation of miR-28-5p in Glioma Proliferation and Invasion

doi: 10.3389/fonc.2019.01413

Figure Lengend Snippet: TRPM7 negatively regulates miR-28-5p in glioma cells. (A) Total RNA from biological triplicate samples, either with scramble control (siCtrl, A423, A424, and A425) or siTRPM7 (A427, A428, and A434), were extracted and then parallelly subjected to human miRNA array assay. In-depth data analysis from miRNA microarray data resulted in a list of 16 downregulated and 10 upregulated miRNAs whose transcripts are statistically significant with fold changes greater than 2 by TRPM7 knock-down. (B,C) Validation of miRNA array by siTRPM7 by qPCR. miR-26b-5p, miR-4530, and miR-28-5p were validated by real-time miRNA quantification using stem-loop RT-PCR in glioma cell lines A172 (B) and SNB19 (C) . A set of endogenous RNAs SNOD48, SNOD44, SNOD47, and U6 served as endogenous controls in real-time miRNA quantification to normalize miRNA gene expression. (D) Validation of miRNA array by overexpressing TRPM7. The constructs of wt hTRPM7 (M7-WT), Δ kinase (M7-DK), K1648R (MT-KR), and control (Ctrl) were introduced into and expressed in glioma cell lines A172, U373MG, SNB19, and U87MG for 48 h. Transfection efficiency was determined by TRPM7 mRNA expression detected by qRT-PCR. GAPDH was used as an internal control to monitor the presence of amplified cDNA in samples (upper left). Western blots by overexpression of individual TRPM7 constructs confirmed the data from qRT-PCR, which showed significantly increased TRPM7 in M7-WT, M7-KR, and M7-DK compared to the control ( D , right). The levels of miR-28-5p were determined by stem-loop RT-PCR (lower left). All the data were represented as fold changes relative to the appropriate controls and are the mean ± SD of triplicated samples performed in three independent experiments. (* P < 0.05, ** P < 0.01).

Article Snippet: Then, total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA) and was subjected to the human miRNA array assay by LC Sciences (Houston, TX, www.LCsciences.com ) using MRA-1001B2 version miRHuman 22, containing 2,632 standard mature miRNA unique probes with 3 repeats, 50 controls (8–32 repeats) based on Sanger miRBase Release 22 ( http://www.mirbase.org/ ).

Techniques: Control, Microarray, Knockdown, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Construct, Transfection, Expressing, Quantitative RT-PCR, Amplification, Western Blot, Over Expression

Journal: Expert Reviews in Molecular Medicine

Article Title: Review of microRNA detection workflows from liquid biopsy for disease diagnostics

doi: 10.1017/erm.2025.2

Figure Lengend Snippet: Overview of unique miRNA detection workflow for targeted disease diagnostics from liquid biopsies

Article Snippet: Thermal cycling amplification - Stem-loop RT-qPCR , Fluorescence – SYBR Green/ Probe based , Tissue, Cells , Spin column , miRNeasy Mini Kit (Qiagen) , miRCURY LNA RT kit + miRCURY LNA SYBR Green PCR Kits/ miRCURY LNA miRNA PCR Assays (Qiagen) , Custom , Custom , Target dependent , Qiagen.

Techniques: Isolation, Amplification, Fluorescence, SYBR Green Assay, Clinical Proteomics, Purification, Bacteria, Virus, Extraction, Infection, Nucleic Acid Electrophoresis, Electrophoresis, cDNA Synthesis, Imaging, CRISPR, Centrifugation, Electrochemiluminescence, RNA Sequencing, Next-Generation Sequencing, Flow Cytometry, Microarray, Magnetic Beads

Journal: Expert Reviews in Molecular Medicine

Article Title: Review of microRNA detection workflows from liquid biopsy for disease diagnostics

doi: 10.1017/erm.2025.2

Figure Lengend Snippet: Schematic illustration of thermal amplification methods for miRNA detection. A. Stem-Loop RT-qPCR (Refs , , , , ) with (i) SYBR Green detection - universal and (ii) Probe-based detection - specific; B. Poly-A Tail RT-qPCR; (Refs , ) C. Sensitive and Multiplexed One-Step RT-qPCR (SMOS-qPCR); (Ref. ) and D. Droplet digital PCR (ddPCR). (Refs , )

Article Snippet: Thermal cycling amplification - Stem-loop RT-qPCR , Fluorescence – SYBR Green/ Probe based , Tissue, Cells , Spin column , miRNeasy Mini Kit (Qiagen) , miRCURY LNA RT kit + miRCURY LNA SYBR Green PCR Kits/ miRCURY LNA miRNA PCR Assays (Qiagen) , Custom , Custom , Target dependent , Qiagen.

Techniques: Amplification, Quantitative RT-PCR, SYBR Green Assay, Digital PCR

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long noncoding RNA MPRL promotes mitochondrial fission and cisplatin chemosensitivity via disruption of pre-miRNA processing

doi: 10.1158/1078-0432.CCR-18-2739

Figure Lengend Snippet: (A) Schematic flowchart depicting the strategy for microarray analysis and validation of lncRNAs. (B) Scatter plot of lncRNA expression in TSCC cells with or without cisplatin treatment. The red dots indicate differentially expressed lncRNAs (fold change ≥2); black dots indicate a fold change less than two in both cell lines, while green dots and blue dots indicate fold changes less than two in the SCC-9 and CAL-27 cells, respectively. X and y-axes, normalized sample signal values (log2 scale). (C) Thirty-eight lncRNAs upregulated by cisplatin treatment were identified by qRT-PCR in both CAL-27 and SCC-9 cells. (D) Eleven lncRNAs were significantly upregulated in TSCC tumors from chemosensitive and nonchemosensitive patients. (E) Schematic annotation of the MPRL genomic locus on chr5:43,016,152–43,019,003 in humans. Green rectangles represent exons. (F) The expression of five splice variants of LOC648987 in CAL-27 and SCC-9 cells. (G) Representative images (MRI scanning) of tumor response (upper panels) and MPRL expression (lower panels) in tissue specimens from patients with chemosensitive (S) and nonchemosensitive (NS) tumors. Scale bar, 20 μm. (H) Prediction of TFs of MPRL by overlaying ChIP-seq data from UCSC and the virtual laboratory of PROMO. (I) Binding motif analysis showing enriched E2F1 motifs in the MPRL promoter; the arrow indicates the binding sites with the highest score. The green to red color gradation is based on the ranking of each binding site from the minimum (green) to maximum (red) scores, which were analyzed by JASPAR. (J) ChIP-qPCR analysis of the E2F1 genomic occupancy in the MPRL promoter in CAL-27 and SCC-9 cells, as indicated. (K) Luciferase assay showing that E2F1 knockdown inhibits MPRL promoter activity in CAL-27 cells. (L) Cisplatin induced upregulation of E2F1 and MPRL expression, while silencing E2F1 decreased MPRL levels in both TSCC cell lines. #P<0.05, *P<0.01 and **P<0.001 versus control, 2-tailed Student’s t tests (D and J); **P<0.001, 1-way ANOVA followed by Dunnett’s tests (K and L).

Article Snippet: Sample labeling and array hybridization were performed by Arraystar Human lncRNA Microarray V3.0 according to the Arraystar microarray-based gene expression analysis protocol (Arraystar, Inc., Rockville, MD, USA). miRNA expression profiles CAL-27 and SCC-9 cells stably transduced with shRNA targeting MPRL were treated with cisplatin at the IC50 (Sigma, USA) for 24 h for miRNA microarray assays.

Techniques: Microarray, Biomarker Discovery, Expressing, Quantitative RT-PCR, ChIP-sequencing, Binding Assay, ChIP-qPCR, Luciferase, Knockdown, Activity Assay, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long noncoding RNA MPRL promotes mitochondrial fission and cisplatin chemosensitivity via disruption of pre-miRNA processing

doi: 10.1158/1078-0432.CCR-18-2739

Figure Lengend Snippet: (A-D) Knockdown of MPRL attenuated mitochondrial fission and apoptosis in CAL-27 and SCC-9 cells. Mitochondrial fission was detected by staining with MitoTracker Red (left panel) and quantified (right) (A); Scale bar, 3 μm; cell apoptosis was detected using flow cytometry (B), TUNEL (C), and caspase-3/7 activity assays (D). (E) Target miRNAs of MPRL were screened by microarray in cells treated with cisplatin. Heat map (left panel) and Venn diagrams (right) depicted differentially expressed miRNAs in cisplatin-treated CAL-27 and SCC-9 cells stably expressing shMPRLs (fold change ≥1.5). (F) The inhibitory effect of MPRL knockdown on mitochondrial fission was attenuated after inhibiting miR-483–5p levels. Mitochondrial fission was detected by staining with MitoTracker Red. (G) Western blot analysis showed that the inhibitory effect of MPRL knockdown on FIS1 expression was attenuated after inhibiting miR-483–5p levels. *P<0.01 and **P<0.001, 2-tailed Student’s t tests.

Article Snippet: Sample labeling and array hybridization were performed by Arraystar Human lncRNA Microarray V3.0 according to the Arraystar microarray-based gene expression analysis protocol (Arraystar, Inc., Rockville, MD, USA). miRNA expression profiles CAL-27 and SCC-9 cells stably transduced with shRNA targeting MPRL were treated with cisplatin at the IC50 (Sigma, USA) for 24 h for miRNA microarray assays.

Techniques: Knockdown, Staining, Flow Cytometry, TUNEL Assay, Activity Assay, Microarray, Stable Transfection, Expressing, Western Blot