rt 2 pcr primer sets for human xt-i, aggrecan, il-1r1, tgf-β1 and ribosomal protein s29  (SuperArray Bioscience Corporation)

Journal: PLoS ONE

Article Title: Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

doi: 10.1371/journal.pone.0139489

Figure Lengend Snippet: (A) SKOV-3 or (B) OVCAR-3 cells were treated with lysophosphatidic acid (LPA, 5 μM) for 0.5, 1 or 2 h prior to the assessment of ATF3 and miR-30c-2-3p expression. The data is presented as a line graph of the relative ratio. (C) Cells were transfected with miRNA scramble or miR-30c-2-3p for 24 h prior to treatment with lysophosphatidic acid (LPA, 5 μM) for the times indicated and assessment of ATF3 expression. The bar graph shows the average relative ratio normalized to a housekeeping control. ***p<0.001, *p<0.05 (D) SKOV-3 cells were transfected with miR-30c-2-3p or a scramble miRNA control for 48 h prior to the quantification of mRNA transcripts. (E) SKOV-3 or (F) OVCAR-3 cells stably expressing an ATF3 3´UTR encoded luciferase vector (wild type) or a vector with mutations in the 3´UTR sequence (mutated) were transfected with either miR-30c-2-3p, anti-miR-30c-2-3p (anti-miR), miR-30c-2-3p + anti-miR-30c-2-3p or a control (negative-target miRNA) for 48 h prior to luciferase signal intensity measurement. ***p<0.001.

Article Snippet: Quantitative RT-PCR Taqman primer set for miR-30c-2-3p was purchased from Life Technologies.

Techniques: Expressing, Transfection, Stable Transfection, Luciferase, Plasmid Preparation, Sequencing

Journal: PLoS ONE

Article Title: Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

doi: 10.1371/journal.pone.0139489

Figure Lengend Snippet: (A) SKOV-3 cells were transfected with either non-targeting-miRNA control or anti-miR-30c-2-3p, prior to treatment with LPA (5 μM) for the times indicated and visualization of ATF3 protein bands. (B) SKOV-3 cells were transfected with anti-miR-30c-2-3p for 48 h and then treated with lysophosphatidic acid for the times indicated prior to measuring ATF3 mRNA expression using qRT-PCR, in comparison to non-targeting-miRNA control. ***p<0.001 (C) ATF3 protein was measured among unstimulated SKOV-3 cells transfected for 48 h with either miR-30c-2-3p, anti-miR-30c-2-3p or a negative, non-targeting-miRNA control.

Article Snippet: Quantitative RT-PCR Taqman primer set for miR-30c-2-3p was purchased from Life Technologies.

Techniques: Transfection, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

doi: 10.1371/journal.pone.0139489

Figure Lengend Snippet: (A) SKOV-3 cells were treated with increasing concentrations of lysophosphatidic acid (LPA) as indicated in μM for 1 h before measuring ATF3 mRNA and miR-30c-2-3p. (B) SKOV-3 cells were processed for immunoblotting to demonstrate the ability of the ATF3 expression vector and siATF3 to modulate ATF3 protein. Similarly, siATF3 enforced expression demonstrates the ability of the siRNA to suppress ATF3 mRNA transcription in SKOV-3 and OVCAR-3 cells using qRT-PCR normalized to microglobulin. (C) OVCAR-3 and SKOV-3 cells were transfected with siATF3 or an siNon-targeting control for 48 h prior to stimulation with LPA (5 μM, 1 h), followed by quantification of miR-30c-2-3p. (D) OVCAR-3 and SKOV-3 cells were transfected with an ATF3 expression vector or a control vector for 48 h prior to quantification of miR-30c-2-3p. ***p<0.001.

Article Snippet: Quantitative RT-PCR Taqman primer set for miR-30c-2-3p was purchased from Life Technologies.

Techniques: Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR, Transfection

Journal: PLoS ONE

Article Title: Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

doi: 10.1371/journal.pone.0139489

Figure Lengend Snippet: (A) ATF3 mRNA expression was measured in SKOV-3 cells after 24, 48 and 72 h of transfection with an ATF3 expression vector. (B) Untreated SKOV-3 cells were transfected as indicated with a combination of either ATF3 expression vector, control vector or anti-miR-30c-2-3p, and then stained for fluorescence imaging using a whole cell marker. (C) High-throughput, automatic quantification software counted ~10 fields of SKOV-3 cells (equivalent to 150–750 cells in total) from the previous experiment. *p<0.05, comparing ATF3 expression vs. control, untreated or control + negative-miR conditions. (D) Cell viability was measured in SKOV-3 cells after ATF3 over-expression compared with control vector after treatment with the indicated concentrations of camptothecin. ***p<0.001.

Article Snippet: Quantitative RT-PCR Taqman primer set for miR-30c-2-3p was purchased from Life Technologies.

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Fluorescence, Imaging, Marker, High Throughput Screening Assay, Software, Over Expression

Journal: PLoS ONE

Article Title: Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p

doi: 10.1371/journal.pone.0139489

Figure Lengend Snippet: (1) Receptor signaling mediated by lysophosphatidic acid (LPA) (2) induces ATF3 expression. (3) The expression of pre-miR-30c-2 also increases, along with ATF3. (4) Processing leads to miR-30c-2-3p transcripts and subsequent binding to ATF3, causing silencing of further ATF3 expression. (Note: model does not accurately depict proper localization of molecules.)

Article Snippet: Quantitative RT-PCR Taqman primer set for miR-30c-2-3p was purchased from Life Technologies.

Techniques: Expressing, Binding Assay

Journal: Oncology Letters

Article Title: Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice

doi: 10.3892/ol.2013.1526

Figure Lengend Snippet: Expression of Foxp3 mRNA. Foxp3 mRNA in B16F10 melanoma cancer cells was analyzed by quantitative (q)PCR and normalized to β-actin as an endogenous control and murine lymphocytes as a positive control. The data represent the mean number from three repeat samples ( * P<0.005 vs. lymphocyte control). Foxp3, forkhead box P3.

Article Snippet: After total RNA isolation from the tumor cell line B16F10 and reverse transcription into cDNA, qPCR was performed using the Chromo4™ Real-Time PCR Detector (Bio-Rad, Hercules, CA, USA), the SYBR Supermix kit (Invitrogen, Paisley, UK), the RT 2 PCR Primer Set for Foxp3 (SuperArray Biosciences, Frederick, MD, USA) and β-actin as a reference gene (RT 2 PCR Primer Set; SuperArray Biosciences).

Techniques: Expressing, Positive Control