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Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs.

ABSTRACT: Changes in gene expression are commonly assessed relative to the expression of housekeeping genes, which are assumed to remain unchanged. We tested this assumption in cerebral arteries obtained from dogs 4 and 7 days after subarachnoid hemorrhage (SAH) had been created using the double hemorrhage model. Basilar arteries were removed and examined for expression of messenger ribonucleic acid (mRNA) levels using quantitative real-time polymerase chain reaction. Cross-sections of basilar arteries were stained immunohistochemically for proliferating cell nuclear antigen (PCNA) and 4',6-diamidino-2-phenylindole (DAPI). Positively stained cells were counted and numbers obtained were normalized to the cross-sectional area. The results were compared to normal dog basilar arteries contracted pharmacologically in vitro. SAH resulted in significant vasospasm (P<0.001 for each, paired t-tests). There were significant increases in mRNA for beta-actin (441%, P=0.01), glyceraldehyde-3-phosphate dehydrogenase (566%, P=0.007) and 18S ribosomal RNA (320%, P=0.025) 7 days after SAH. Total mRNA was increased 7 days after SAH relative to genomic DNA (157%, P=0.009). There were significant increases in the number of cells in the tunica media and adventitia of arteries after SAH and a significant decrease in the media after contraction in vitro. Cells in the tunica media and adventitia labeled with PCNA were significantly increased at both times after SAH. Transcripts for housekeeping genes are increased after SAH, making standardization to them potentially invalid. The increase is due to proliferation of cells in the adventitia and increased total mRNA in the media and adventitia.

SUBMITTER: Aihara Y

PROVIDER: S-EPMC2478696 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs.

Aihara Yasuo Y Jahromi Babak S BS Yassari Reza R Takahashi Masataka M Macdonald R Loch RL

Journal of neuroscience methods 20080410 1

Changes in gene expression are commonly assessed relative to the expression of housekeeping genes, which are assumed to remain unchanged. We tested this assumption in cerebral arteries obtained from dogs 4 and 7 days after subarachnoid hemorrhage (SAH) had been created using the double hemorrhage model. Basilar arteries were removed and examined for expression of messenger ribonucleic acid (mRNA) levels using quantitative real-time polymerase chain reaction. Cross-sections of basilar arteries we ...[more]

PMID: 18490059

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Project description:The diagnosis of cerebral vasospasm after Subarachnoid-Hemorrhage is currently very difficult, additional tools such as blood biomarkers are necessary. We tested the ability of gene expression profiles of blood cells to predict vasospasm. 32 patients suffering subarachnoid-hemorrhage were included in this prospective monocentre study. They were grouped according to have a complicated cerebral vasospasm (Vasospasm) or not (Control) and Paired according to age (+/- 10 years), sex, Fisher grade (+/- 1), location, smoking (at least 3 first parameters). Gene expression profiles of blood cells were determined using 25,000~gene microarray. Blood sample: 2.5 mL harvested in PAXgene® Blood RNA tubes (PreAnalytix) RNA extraction: PAXgene® Blood RNA kit (Qiagen). We used a Universel Reference RNA (Stratagene). RNA amplification and labelling: kit Amino Allyl MessageAmp II (Ambion). We hybridized 4 microarrays per patient using pangenomic microarrays from the "Réseau National des Génopôles" (Illkirch, France). 2 slides were hybridized with reference RNA labelled Cy3 and patient RNA labelled Cy5, and 2 slides were hybridized with reference RNA labelled Cy5 and patient RNA labelled Cy3. Hybridation : Agilent protocol with few modifications : 750 ng of each labelled RNA were hubriddized at 60°C during 17 hours in an Aglient hybridization oven. After washings, Slides were scanned with a GenePix 4000B scanner (Molecular Devices). Image intensity data were extracted with GenePix Pro 6.0 analysis software. Quantification of Cy3 and Cy5 and selection of good spots were performed using the MAIA software (Novikov E and Barillot E. Software package for automatic microarray image analysis (MAIA). The ACUITY software was then used to normalize log ratios Cy3/Cy5 with Lowess non linear normalization, to filter out genes not present in at least 3 slides out of 4, to evaluate the reproducibility of the 4 microarrays of each patient (hierarchical clustering, Self Organizing Maps). Statistical analyses to insure reproducibility was performed using Excel (correlation coefficients, ANOVA). Only slides that passed all reprocubility tests were validated.

Dataset Information

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs.

Publications

Induction of housekeeping gene expression after subarachnoid hemorrhage in dogs.

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