Transcription profiling of human brain samples to investigate damage caused by AIDS infection of the CNS

ABSTRACT: This project will help us to have a more accurate and useful explanation at molecular level, of the damage caused by HIV when it infects the central nervous sythem (CNS) in Humans. CNS dysfunction is an important cause of morbidity and mortality in patients with human immunodeficiency virus-I (HIV-1) infection and the acquired immunodeficiency virus syndrome (AIDS). Minor cognitive/motor dysfunction (MCMD) and HIV-1-associated dementia (HAD) develop in the absence of opportunistic infections and lymphoma, cerebrovascular disease and metabolic disorders. Patients with HAD may or may not present an associated HIV encephalitis with viral replication limited to cells of monocyte origin. The inflammatory infiltrates of HIVE include activated microglia and perivascular and parenchymal monocytes, multinucleated giant cells and lymphocytes. In addition, HIVE may be accompanied by significant loss of neurons, presynaptic terminals and abnormal dendrites. To determine if altered expression of neuronal growth factor, chemokine and cell death genes identified possible mechanisms of brain injury in AIDS patients with and without HIVE; Changes of gene expression occurring in brain of AIDS patients with and without HIVE, induce development of brain atrophy, mild gliosis, enhanced vascular permeability, along with alterations in neuronal HIV-1 chemokine coreceptors. In the cases of HIVE more remarkable gene expression changes result in significant loss of neurons, presynaptic terminals and abnormal dendrites. All these changes can be evaluated by microarray gene expression analysis and validated by Q-RT-PCR; The procedure includes 10 steps: 1-Frontal Cortex dissection from fresh frozen autopsy human brain samples (fragments of 0.8-1 cm3 are used) 2-totalRNA extraction using RNeasy Maxi Kit and following hand book protocol; Quiagen Inc., 3-RNA quantity determination using the NanoDrop ND-1000 UV-Vis Spectrophotometer: Nanodrop-Technologies 4-RNA quality analyzed by ribosomal fractions 28s/18s ratio, using by Agilent RNA 6000 Assay Microchips: Agilent Technologies 5-mRNA suitability by RT-PCR test for housekeeping genes and genes of interest. 6-mRNA amplification and labeling in two steps: a-Generation of T7-conatining double stranded cDNA using T7-Poly-T Strategy (Invitrogen) b-mRNA amplification and labeling (aRNA) by in vitro transcription using T7 RNA polymerase, T7-cDNA as template and biotin-labeled ribonucleotides 7-Biotin-labeled aRNA quantity assess, as in step 3 8-Biotin-labeled aRNA determination by amplified RNA electropherogram. Agilent RNA 6000 Assay Microchips: Agilent Technologies 9-Biotin-labeled aRNA fractionation, hybridization onto HG-95aVer2 oligonucleotide array and scanning, using Affymetrix platform: Affymetrix 10-Statistical gene expression analysis: Using GeneSpring Software (Slicon Gnetics) a-Normalization to the 50th percentile and per gene to specific control samples, using 'Disease vs. Normal' as the main parameter. b-Fold of expression filter (2 folds cutoff) using signal data in ratio mode interpretation c-Welch t-test, with p< 0.05 for significance, using log of ratio mode interpretation d-Differentially expressed genes clustering by K-means, experiment and gene trees e-Differentially expressed genes analysis and classes classification by Gene Ontology and GeneMapp pathways

ORGANISM(S): Homo sapiens

SUBMITTER: Elizabeth Salomon 

PROVIDER: E-GEOD-4755 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress