Project description:We performed whole genome microarrays on selected cell lines in order to identify transcriptionally regulated target genes of selected miRNAs.
Project description:IGR39 cells, seeded at a density of 6x104 cells/well in 12-well culture plates, were transfected with 5 nM of miR-211 mimic or negative control (NCM) as described in the main text. Samples for total RNA extraction were collected 48h after transfection. RNA quality was assessed using RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer (Agilent, Paolo Alto, USA). Gene expression profiling experiments using GeneChipM-. Human Gene 2.0 ST arrays and miRNA profiling using GeneChipM-. miRNA arrays (Affymetrix, Santa Clara, CA, USA) were performed on two independent samples at the CRP-SantM-i Microarray Unit (Luxembourg) as described before (Reinsbach et al., 2012) and according to standard protocols. Standard pipeline from the PartekM-. Genomics SuiteTM (Partek GS) software was used for analysis of data files. Lists of genes were generated by pair-wise comparison of expression data sets (negative control vs. mimic treated samples at 48h).
Project description:We used the autophagy microarray to profile the transcriptome of well-characterized TNF sensitive MCF-7 cell line and corresponding TNF-resistant 1001 clone.
Project description:HIV-1 and HIV-2 are two etiological agents of Acquired Immune Deficiency Syndrome (AIDS). Several differences exist between these two retroviruses in terms of geographical distribution, replication, transmission and progression to AIDS. The molecular reasons explaining these features are largely unknown. One reason could rely on host factors able to differently counteract HIV replication. Among these factors, cellular microRNAs (miRNAs) have recently emerged as playing crucial roles. One aspect of the complex interplay between HIV and host miRNAs is the ability of HIV-1 to modulate host miRNAs and thereby to create favorable conditions for its replication. Here, we sought to compare the miRNA modulations elicited by HIV-1 and HIV-2 using an unbiased experimental strategy based on miRNA arrays. Surprisingly, we observed that these two unrelated HIVs similarly modulated the host miRNA repertoire, when utilizing CD4 and CXCR4 for entry. However, these modulations were different from the changes triggered by HIV-1 using CD4 and CCR5. In accordance with the mode of action of miRNAs, our observations were confirmed at the mRNA level. We concluded that co-receptor utilization (CXCR4 or CCR5), as opposed to genomic organization and phylogeny, is a key event determining the modulations of the host miRNA repertoire.
Project description:Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. The microRNA (miRNA) isolation from human skin tissue was performed using miRNeasy FFPE kit (Qiagen). For PCR array, miRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of miRNAs from 3 normal skins, 3 SSc, 3 LSc or 3 keloid were prepared, and miRNA expression profile in each disease in vivo was evaluated using RT2 Profiler PCR Array. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into 96-well RT2 miRNA PCR Array that includes primer pairs for 88 human miRNAs (SABiosciences). Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. Control donors were each matched with diseases for age, sex, and biopsy site.
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
Project description:Ischemic preconditioning (4 cycles of 5 min ischemia and 5 min reperfusion) with a final reperfusion time of 120 minutes was performed in C57BL/6N (The Jackson Laboratory) or Per2-/- mice. Heart tissue was snap-frozen with clamps pre-cooled to the temperature of liquid nitrogen. Micro RNA was isolated with Trizol (Invitrogen) and purified using RT2 qPCR-Grade miRNA Isolation Kit (SABiosciences-Qiagen). cDNA template was generated using RT2 miRNA First Strand Kit (SABiosciences-Qiagen). miRNA expression was performed using RT2 miRNA PCR Array Mouse miFinder (SABiosciences-Qiagen).
Project description:Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.
Project description:This study evaluated changes in gene expression upon IL-7 treatment in human naive and memory Treg. CD4+CD25+CD127low naïve and memory Treg were isolated from fresh PBMC and separately stimulated with ?CD3?/CD28 coupled beads (Invitrogen-Dynal) at a 1:10 bead/T cell ratio, treated with or without 10 ng/ml of rhIL-7 (R&D Systems) for 16 hours. qPCR gene expression profiling of CD4+CD25+CD127low naïve and memory Treg obtained from 2 separate donors. Cell lysates were prepared separately from the 2 donors and pooled prior to RNA extraction.