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RNA expression profiling of CD13/CD33-positive and CD13/CD33-negative B-ALL patients [mRNA expression]

ABSTRACT: To study the effect of expression of CD13/CD33 on the pathogenesis of B-cell acute lymphoblastic (B-ALL), we collected samples from 4 CD13/CD33-positive and 4 CD13/CD33-negative B-ALL patients and performed RNA-seq.

ORGANISM(S): Homo sapiens

PROVIDER: GSE197178 | GEO | 2023/12/31

REPOSITORIES: GEO

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Project description:The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To directly address the function of myeloid CD13 we created a CD13 null mouse and assessed the responses of purified primary macrophages or dendritic cells from wild type and CD13 null animals in cell assays and inflammatory disease models where CD13 has been previously implicated. We find that mice lacking CD13 develop normally with normal hematopoietic profiles. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation and antigen presentation that we tested, but may contribute to adhesion to endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 wild type and null macrophages argue against compensatory mechanisms. Analysis of the dataset with Ingenuity Pathway Analysis software did not suggest that loss of CD13 resulted in a purturbation of any specific biological pathways, processes or networks. Therefore, while CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of both normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis or myeloid cell function. Method: RNA was isolated from resting peritoneal or bone marrow derived macrophages from wild type or CD13 null mice using Trizol according to the manufacturer’s protocol. For quality control, RNA purity and integrity was evaluated by denaturing gel electrophoresis, OD 260/280 ratio and analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was amplified and purified using the Ambion Ilumina RNA amplification kit (Ambion, www.ambion.com). Briefly, 300 ng of total RNA was reverse transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed and labeled with biotin-16-UTP. The labeled cRNA was analyzed on the Agilent 2100 Bioanalyzer and quantitated by Nanodrop analysis (www.nanodrop.com). The labeled cRNAs were hybridized to the Mouse WholeGenome- 6 BeadChip (Illumina Inc.,) for 16 hours at 58°C, as per manufacturer's instructions. The BeadChips were washed, stained with streptavidin-Cy3 and scanned on the Illumina BeadArray Reader. Analysis: Microarray data was extracted, normalized and analyzed using Illumina GenomeStudio software. Illumina MouseWG-6 v 2.0 Expression BeadChip (Illumina, San Diego, CA) contains 50-mer gene specific oligonucleotide probes corresponding to 45,281 mouse transcript variants. There is on average a 30-fold redundancy for each transcript per array. Intensity data were normalized using the Quantile algorithm in GenomeStudio. Differential expression of each gene, relative to the respective control was evaluated using the Illumina custom error model that calculates a p value as a function of intensity, differential and biological, technical and nonspecific variation. Those samples with p values less than 0.05 were deleted, the relative expression of CD13 null vs. wild type calculated and expressed as fold wild type, and the data ranked from low to high expression relative to wild type values. The dataset was further analyzed using the Ingenuity Pathway Analysis software (Ingenuity Systems, http://www.ingenuity.com).

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RNA expression profiling of CD13/CD33-positive and CD13/CD33-negative B-ALL patients [mRNA expression]

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