Unknown,Transcriptomics,Genomics,Proteomics

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Expression analysis of effect of Intestine-specific knockdown of KLF5 on radiation induced intestinal injury

ABSTRACT: The objective was to determine the role of Krüppel-like factor 5 (KLF5) in radiation-induced intestinal injury. Mice with intestinal-specific knockdown of KLF5 (Cre+ mice) were generated and their response to radiation was compared with controls (Cre- mice). Mice were given 15 Gy total body irradiation (TBI). The mice intestines were harvested at 6h post TBI and screened for differentially expressed genes by microarray analysis. We identified 11,004 and 2,466 differentially expressed genes in non-irradiated and irradiated mice at 6h post TBI, respectively. KLF5 knockdown down-regulated genes related to DNA damage repair pathways such as nucleotide excision repair, mismatch repair, non-homologous end joining and the Fanconi anemia pathway, which may suggest a novel function of KLF5. A four chip study using total RNA recovered from four pooled intestine tissues of four experimental groups. The four experimental groups respectively were the 6h-cre+ group, 6h-cre- group, con-cre+ group and con-cre- group. Equal mass amounts of total RNA were pooled from each small intestine to yield a sample representing RNA from 3 separate mice in each group. One pooled sample in every group were tested with one chip. Each chip measures the expression level of 44,170 genes from C57BL/6 mouse (background) with three 60-mer probe pairs per gene.

ORGANISM(S): Mus musculus

SUBMITTER: Ming Li

PROVIDER: E-GEOD-66655 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common form of leukemia in children. Multiple lncRNAs participate in normal and may be implicated in malignant hematopoiesis associated with blood cell cancers, such as leukemia. Currently, the expression profile of lncRNAs in pediatric AML is unclear. In this study, we evaluated the lncRNA expression profile in the tissue of three pediatric AML patients with lncRNA microarray techniques. In order to gain insight into the function of targets of lncRNAs, GO term and KEGG pathway annotation were applied to the target gene pool. qPCR was performed to evaluate the expression patterns of dys-regulated lncRNAs. Bone marrow specimens were obtained at the time of diagnosis during routine clinical assessment of 3 pediatric patients with AML, who presented at the Department of Hematology and Oncology, Children's Hospital of Soochow University between 2000 and 2011. Additionally, bone marrow samples from 3 healthy donors were analyzed as controls. Human LncRNA Array analysis was performed by KangChen Bio-tech, Shanghai P.R. China. Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. For microarray analysis, Agilent Array platform was employed. The sample preparation and microarray hybridization were performed based on the manufacturerâs standard protocols with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLYâ¢ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3â bias utilizing a random priming method. The labeled cRNAs were hybridized onto the Human LncRNA Array v2.0 (8 x 60K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 4 out of 6 samples have flags in Present or Marginal (âAll Targets Valueâ) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Pathway analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show the distinguishable LncRNAs and mRNAs expression pattern among samples.

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Expression analysis of effect of Intestine-specific knockdown of KLF5 on radiation induced intestinal injury

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