View Project torre-affy-human-217240

Project Summary
Status:	Public
Publications:	2 Published
Project Detail		Data Detail
Platform:	Affymetrix	MIAME Areas	Compliance
Species:	Human	Array Design Detail	false
Organ/Tissue Type:	brain	Experiment Detail	true
Organ Region:	frontal cortex	Sample Detail	false
Cell Type:	SH-EP	Hybridization Detail	false
Study Type:	subclassification	Measurement Detail	false
Disease/Condition:	HIV-associated dementia
Replicates:	4
Expected Samples:

Available Actions

Investigator Contact Detail
Name	Jorge E Torres-Munoz
Street Address:	1550 NW 10th Ave PAP-429
City, State/Province:	MIami , FL
Zip/Postal Code:	33136
Country:	United States
Work Phone:	(305) 243 3824
Fax:	(305) 243 4086
E-mail:	jtorres@med.miami.edu

Proposal Detail

Grant:

303017

Status:

Public

Service Type:

Start to Finish Profiling

IRB:

2002-0613A

IRB date:

2004-06-09

Study Relevance:

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.

Hypothesis:

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

Specific Aim:

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

Experimental Procedure and Design:

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

Quality Control Description:

Case Concentration 280/260 ratio 28s/18s ratio A01-044 395.4 ng/ul 2.06 0.7 D03-0046 861.2 ng/ul 2.01 0.9 DME01-1991 227.5 ng/ul 2.01 1 DME02-0053 475.8 ng/ul 2.0 1

Quality Control Types:

biological_replicate

Replicate Description:

Cases A01-044 and D03-0046 are biological replicas: HIVE Cases DME01-1991 and DME02-0053 are bilogical replicas: AIDS cases without encephalitis Scanning results from these bilogical replicas will be added to a set of microarray analysis results, previously obtained in our laboratory

Replicate Types:

biological_replicate

Experimental Factors:

Conditions that are tested in the experiment. At least one is required. Experimental factors are the independent variables in the experiment.

Factor Name	Description	Factor Category
Normal HIV negative, AIDS with Normal Brain and AIDS with HIVE	Our whole experiment design include ...	disease_staging

Project Samples

Samples associated with this project.

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Name	Description	Bio-Source	Extracts
sample A01-044	Sample A00-44 correspond to...	A01-044	1
sample D03-0046	Sample D03-0046 correspon...	D03-0046	1
sample DME01-1991	Sample DME01-1991 correspon...	DME01-1991	1
sample DME02-0053	Sample DME02-0053 correspon...	DME02-0053	1

Project Hybridizations

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Name	Array	Labeled Extract
HG_U95Av2_1_hyb	HG_U95Av2_1	sample A01-044 extraction_le1
HG_U95Av2_2_hyb	HG_U95Av2_2	sample D03-0046 extraction_le1
HG_U95Av2_3_hyb	HG_U95Av2_3	sample DME01-1991 extraction_le1
HG_U95Av2_4_hyb	HG_U95Av2_4	sample DME02-0053 extraction_le1
Hybridization8		sample A01-044 extraction_le1
Hybridization9		sample D03-0046 extraction_le1
Hybridization10		sample DME01-1991 extraction_le1
Hybridization11		sample DME02-0053 extraction_le1

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