Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of miRNA in mouse models of melanoma comparing niches of tumour tissue to adjacent normal tissue

ABSTRACT: This experiment focused on the miRNA expressions within the tissues specific niches of mice afflicted with melanoma, compared to littermates without malignancy. BRafCA/ PtenloxP/ Tyr::CreERT2 or genetically matched littermates without Cre recombinase were treated with 2µl 4-hydroxytamoxifen (5mM), every other day, for on days 0, 2, and 4, on the shaved right flank of the abdomen of the animal. Mice slowly grow melanoma at the treated site, and some metastases distal to treatment location (eg. Ear or opposite side of abdomen). Endpoint criteria was designated as a Samples were taken when tumors reached 1.5mm diameter. Total RNA was isolated from tumors or tamoxifen treated healthy skin, skin adjacent to the treatment site, or the ear ( representing a metastatic site). Total RNA was prepared with FLAG-TAG kit and miRNA microarray 4.0 was performed.

ORGANISM(S): Mus musculus

SUBMITTER: Robert Zeiser

PROVIDER: E-MTAB-6373 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5M-bM-^@M-^Y-WGGWWW-3M-bM-^@M-^Y with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. A FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which were interrogated by qRT-PCR and correlated with cell culture expression levels. Xenografts. Grafting with A549-luc-C8. Experiments were performed in female SCID-beige mice (Charles River) between 8 and 12 weeks of age. Cells were injected into the left flank area of the animals as suspensions of 25 x 106 mL-1 in RPMI, 200 M-BM-5L per injection. Treatment and tumor proliferation monitoring. Mice were treated following the schedule delineated in treatment protocol. Tumor proliferation was monitored using the XENOGEN imaging device. The animals were anesthetized with 2 5 % isoflurane and subsequently transferred to the imaging chamber, whereupon the isoflurane levels were reduced to 1-2.5 %. The floor of the imager was heated to +37 M-BM-:C to avoid hypothermia. Breathing frequency was monitored and not allowed to drop below 1 s-1, adjusting the isoflurane levels accordingly at all times. Endpoint criteria and euthanasia. Animal endpoint criteria encompassed weight loss of over 15 %, restriction of motor function by the engrafted tumor, dehydration of over 10 % and moribund behavior. Where appropriate, the animals were euthanized by asphyxiation in a CO2 chamber. Tumor tissue harvest. Animals were resected and tumors excised using standard forceps, scissors and surgical blades. The tumors were combined into one sample per condition and mechanically sheared in TRIZOL, employing a specialized device (tissue tearer, model 985370). Total RNA workup was performed following the standard TRIZOL procedure, followed by a DNAse digest.

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Transcription profiling by array of miRNA in mouse models of melanoma comparing niches of tumour tissue to adjacent normal tissue

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