Project description:To functionally characterize the role of miR-17 family in HCC, lentiviral vector-based miR inhibitor TuD was used to inhibit miR-17 family of microRNAs in Hep3B cell line Methods: Hep3B HCC cell line was acquired from American Type Culture Collection (ATCC) and miR-17 TuD or NC TuD expressing lines were generated. Microarray profiling of miR-17 TuD or NC TuD expressing samples was performed using Affymetrix HG-U133Plus2 arrays. Briefly, starting with 1 ug total RNA, amplified biotin-labeled cRNA targets were produced using the Enzo Target Labeling method. Fragmented, biotin-labeled cRNA was hybridized to the Affymetrix GeneChip® HG-U133Plus2 Genome Arrays overnight at 45C for 16 to 18 hours according to the manufacturer’s recommendations. Stain, wash (GeneChip Fluidics Station 450, EukGE-WS2v5_450 script) and scan (GCS 3000 7G with the GeneChip® AutoLoader) were performed according to the manufacturer’s recommendations. Microarray data were GC Robust Multiarray Average (GCRMA) normalized using R and the bioconductor.org package gcrma and Log2 transformed.
Project description:HEK293 cells were transfected with control plasmid (pcDNAI/Neo;Invitrogen) or with the plasmid encoding HCaRG. Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen). Chips HG-U133A: - Labeling protocol: Enzo BioArray HighYield RNA Transcript Labeling Kit. - Hybridization: According to the manufacturer's protocol (Affymetrix). - Scanner: GeneArray 2500. - Normalization: Employing GCOS software, chips were normalized using all probe sets scaling option and target signal at 500. Chips HG-U133 Plus 2.0: - Labeling protocol: GeneChip IVT Labeling Kit. - Hybridization: According to the manufacturer's protocol (Affymetrix). - Scanner: GeneChip Scanner 3000. - Normalization: Employing GCOS software, chips were normalized using all probe sets scaling option and target signal at 500. Keywords: parallel sample
Project description:The identification of new agents that may modulate the progression of cancer cell growth is of great interest. In this regard, dietary agents can be utilized to identify molecular targets to be used as part of a chemopreventive strategy. Walnuts contain several bioactive compounds, including pedunculagin, a polyphenol metabolized by microbiota to form urolithins, namely urolithin A (UA). We performed a genomic analysis to study the effect of UA on androgen-dependent LNCaP prostate adenocarcinoma cells. Cells were incubated with 40µM UA for 24 hours, then, RNA was extracted. RNA samples were processed in the automated Biomek FX System (Beckman Coulter), where aRNA was produced, and labeled with Biotin, purified , fragmented and hybridized onto HG U219-24 Array, using the GeneChip HT 3’IVT Express Kit . Afterwards, the GeneTitan® Multi-Channel (MC) Instrument (Affymetrix) was used to hybridize, wash , stain , and scan the arrays. Microarray results were analyzed using the GeneSpring GX v13.0 software.
Project description:TNF-a is elevated in a variety of inflammatory disease involving epithelial tissues. To better understand the the impact of TNF-a on epithelial biology, 3 epithelial cell lines were cultured with and without TNFa and the transcriptomes compared using were profiled using Affymetrix GeneChip® HG-U133+2 array or HG-U133+PM arrays.
Project description:The evaluation of mycotoxicity of type B trichothecenes using a yeast gene expression comparison analysis. The yeast BY4743 derivative PDR5 mutant was used for this study. The yeast cells were treated with trichothecene mycotoxins, and incubated at 25 degree for two hours, respectively. Total RNA was isolated with commercial kit (FastRNA Pro Red kit, Q-Biogene), and amplified RNA (aRNA) was synthesized with 3'IVT Express kit (Affymetrix). All samples were hybridized with Gene Chip (Yeast Genome 2.0 Array, Affymetrix), then each array chip was scanned by GeneChip Sanner 3000 (Affymetrix).
Project description:The evaluation of zymolyase treatment using yeast gene expression comparison analysis. The yeast BY4743 strain was used for this study. Yeast cells were treated with Zymolyase (final conc. 300 U/ml), and incubated at 37 degree for ten minutes. Digestion process was not provided for control sample. Total RNA was isolated with commercial kit (FastRNA Pro Red kit, Q-Biogene), and amplified RNA (aRNA) was synthesized with 3'IVT Express kit (Affymetrix). All samples were hybridized with Gene Chip (Yeast Genome 2.0 Array, Affymetrix), then each array chip was scanned by GeneChip Sanner 3000 (Affymetrix).