Project description:We implemented an optimized processing, using alternative Chip Description Files (CDFs) and fRMA normalization, which improve the quality of downstream analysis. We profiled 44 FFPE primary breast cancer samples using Affymetrix HG-U133 Plus 2.0 microarray platform after RNA amplification with the Nugen WT-Ovation FFPE System
Project description:The study is designed to identify transcriptional differences of CD45-Ter119-Cxcl12-DsRed+ stromal cells at different develomental stages. 50,000 CD45-Ter119-Cxcl12-DsRed+ stromal cells from freshly prepared livers of E15.5, P5 and P10 Cxcl12DsRed/+ mice were sorted into Trizol by flow cytometry. Total RNA was isolated and amplified with the WT-Ovation Pico RNA Amplification system (Nugen) as the manufacturer’s instructions. Sense strand complementary DNA was generated using the WT-Ovation Exon Module (Nugen). Then, cDNA was fragmented and labelled using FL-Ovation DNA Biotin Module V2 (Nugen). The labelled cDNA was hybridized to Affymetrix Mouse Gene ST 1.0 chips following the manufacturer's instructions.
Project description:The use of Affymetrix U133 2.0 Plus chips on FFPE samples when coupled with a qPCR-based sample pre-assessment step, yielded satisfactory results from the point of view of biological reliability. When compared with the Illumina DASL WG platform, specifically designed for degraded RNA, the data generated with the Affymetrix platform showed a wider interquartile range (1.32 vs 0.57, p<2.2x10-16) suggesting a superior discriminatory power within samples as indicated by the good agreement with the immunohistiochemically derived ER status. FFPE primary breast cancer samples profiled using Illumina DASL WG platform after RNA amplification with the Nugen WT-Ovation FFPE System
Project description:The use of Affymetrix U133 2.0 Plus chips on FFPE samples when coupled with a qPCR-based sample pre-assessment step, yielded satisfactory results from the point of view of biological reliability. When compared with the Illumina DASL WG platform, specifically designed for degraded RNA, the data generated with the Affymetrix platform showed a wider interquartile range (IQR 1.32 vs 0.57, p<2.2x10-16) suggesting a superior discriminatory power within samples as indicated by the good agreement with the immunohistiochemically derived ER status. FFPE primary breast cancer samples profiled using Affymetrix HG-U133 Plus 2.0 microarray platform after RNA amplification with the Nugen WT-Ovation FFPE System The following criteria were considered for a direct comparison of 12 GEPs obtained from Affymetrix and DASL platforms: gene variability as defined by IQR, ESR1 expression in ER status subgroups defined by IHC, distribution of fold changes for predefined ER related genes when comparing ER positive and negative samples.
Project description:We discovered that mice that lack Lsd1 in hematopoietic cells were exhibited increased frequencies of CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs, but completely lacked the lin- c-Kit+ Sca-1- myeloid progenitor compartment. To determine the genes altered by Lsd1-loss, CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs from Lsd1fl/fl and Lsd1fl/fl Mx1Cre mice were FACS-purified to be analyzed by gene expression profiling. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform.
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.