Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Using AmpliSeq we have performed quantitative analysis of 20,803 genes in Negative control precursor-miR (NC-Pre-miR) and pre-miR-17 transfected Rheumatoid arthritis Synovial Fibroblasts (RASFs)

ABSTRACT: Purpose: The goals of this study are to determine the effect of microRNA-17 overexpression on 20,803 human genes in RASFs using Ion ProtonTM System platform. Human RASFs from two RA patients were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared using miRNeasy kit (Qiagen). Total RNA integrity was checked using an Agilent Technologies 2100 Bio analyzer (Santa Clara, CA). 10 ng of high quality RNA was used to make cDNA for amplification with the Ion AmpliSeq Transcriptome Human Gene Expression kit (ThermoFisher Scientific). The cDNA was subjected to 12 cycles of amplification with panel primers and barcoded with adapters as recommended. Resulting sequencing libraries were quantified by qPCR using SYBR FAST master mix from KapaBiosystems (Wilmington, MA). Sets of eight libraries were balanced, pooled and sequencing beads produced on an Ion Chef. Sequencing was performed on an Ion P1 semi-conductor sequencing chip using an Ion Protonâ¢ System (ThermoFisher Scientific, Grand Island, NY). Data was collected and primary analysis performed using Torrent Suite software version 5.0.3. Reads were mapped to the panel and expression values determined. R Software version R-3.2.3 was used to generate heatmap. Among the panel of 20,803 genes, the expression of 15,067 genes as shown in the representative heat map was observed in pre-miR-17 and NC-pre-miR transfected RASFs. A total of 664 significantly modulated genes (301 upregulated and 363 downregulated) using Student âtâ test were further utilized for the IPA analysis. The result of IPA predicted the protein ubiquitin pathway as a major canonical pathway affected by the differentially regulated genes. Interestingly, IPA analysis generated an interactome that showed connectivity among various ubiquitin ligases, NF-ÔB family, AP-1/cJun, 20S and 26S proteasome system. Conclusion: Our results clearly shows the major pathways affected by miR-17 overexpression in RASFs were Protein ubiquitination related. mRNA profiles of pre-miR-17 and NC-pre-miR transfected RASFs were generated by AmpliSeq, in duplicate, using Ion Protonâ¢ System.

ORGANISM(S): Homo sapiens

SUBMITTER: Salahuddin Ahmed

PROVIDER: E-GEOD-83930 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts.

Akhtar Nahid N Singh Anil Kumar AK Ahmed Salahuddin S

Journal of immunology (Baltimore, Md. : 1950) 20160817 6

TNF-α is a major cytokine implicated in rheumatoid arthritis (RA), and its expression is regulated at the transcriptional and posttranscriptional levels. However, the impact of changes in microRNA expression on posttranslational processes involved in TNF-α signaling networks is not well defined in RA. In this study, we evaluated the effect of miR-17, a member of the miR-17-92 cluster, on the TNF-α signaling pathway in human RA synovial fibroblasts (SFs). We demonstrated that miR-17 expression wa ...[more]

PMID: 27534557

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Project description:Purpose: The goals of this study are to determine the effect of microRNA-17 overexpression on 20,803 human genes in RASFs using Ion ProtonTM System platform. Human RASFs from two RA patients were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared using miRNeasy kit (Qiagen). Total RNA integrity was checked using an Agilent Technologies 2100 Bio analyzer (Santa Clara, CA). 10 ng of high quality RNA was used to make cDNA for amplification with the Ion AmpliSeq Transcriptome Human Gene Expression kit (ThermoFisher Scientific). The cDNA was subjected to 12 cycles of amplification with panel primers and barcoded with adapters as recommended. Resulting sequencing libraries were quantified by qPCR using SYBR FAST master mix from KapaBiosystems (Wilmington, MA). Sets of eight libraries were balanced, pooled and sequencing beads produced on an Ion Chef. Sequencing was performed on an Ion P1 semi-conductor sequencing chip using an Ion Proton™ System (ThermoFisher Scientific, Grand Island, NY). Data was collected and primary analysis performed using Torrent Suite software version 5.0.3. Reads were mapped to the panel and expression values determined. R Software version R-3.2.3 was used to generate heatmap. Among the panel of 20,803 genes, the expression of 15,067 genes as shown in the representative heat map was observed in pre-miR-17 and NC-pre-miR transfected RASFs. A total of 664 significantly modulated genes (301 upregulated and 363 downregulated) using Student ‘t’ test were further utilized for the IPA analysis. The result of IPA predicted the protein ubiquitin pathway as a major canonical pathway affected by the differentially regulated genes. Interestingly, IPA analysis generated an interactome that showed connectivity among various ubiquitin ligases, NF-ԟB family, AP-1/cJun, 20S and 26S proteasome system. Conclusion: Our results clearly shows the major pathways affected by miR-17 overexpression in RASFs were Protein ubiquitination related.

Dataset Information

Using AmpliSeq we have performed quantitative analysis of 20,803 genes in Negative control precursor-miR (NC-Pre-miR) and pre-miR-17 transfected Rheumatoid arthritis Synovial Fibroblasts (RASFs)

Publications

MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts.

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