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Series GSE1034

Query DataSets for GSE1034

Status

Public on Feb 11, 2004

Title

Temporal Response of Rat Lung to Hemorrhage

Organism

Rattus norvegicus

Experiment type

Expression profiling by array

Summary

Male Sprague-Dawley rats weighing 280-300 g were anesthetized with 1.75% to 2.5% isofluorane and instrumented with two arterial catheters, one externalized for blood withdrawal and the other (not externalized) attached to the Data Sciences International (DSI) PhysioTel (St. Paul, MN, model C50-PXT) monitoring device placed intraperitoneally and used in monitoring blood pressure, temperature, and electrocardiogram data via telemetry. After 7 to 10 days of recovery, and only after the animals had begun to gain weight, the experiment was performed. At the beginning of hemorrhage (H), blood was withdrawn manually through the externalized catheter with 1-ml syringes at l ml/min. Total blood volume was estimated to be 6.5% of the body weight and 40% of this (2.6 ml/100g) was removed to effect the hemorrhage.
Control animals (Con) were uninstrumented and were not hemorrhaged or manipulated in any way. Sham (Sh) rats were instrumented, but not hemorrhaged, and physiologic measures were taken for 4 – 23 hrs 6 hrs prior to euthanasia. At 1, 3, 6, 16, 24, 48, or 72 hours (H) after hemorrhage animals were deeply anesthetized with Nembutal (50 mg/kg), and the post hemorrhage blood sample was collected by cardiac puncture and the animals were euthanized by exsanguination. All tissues were collected within 10 minutes of death and 100 mg amounts of the following tissues were removed and placed in RNALater™ (Ambion, Austin, TX): intestine, liver, lung, kidney, spleen, heart, skeletal muscle (gastrocnemius) skin, and brain.
Total RNA was isolated from each tissue from each animal with 2 rounds of TRI Reagent® (MRC, Cincinnati, OH) and its quality analyzed by gel electrophoresis. A reference preparation consisting of equal amounts of RNA pooled from 10 organs (liver, lung, kidney, spleen, heart, skeletal muscle, skin, jejunum, and brain) of untreated control animals was used as reference RNA. Five-µg samples exhibiting undegraded RNA from each rat lung were reverse transcribed with Superscript II™ (Invitrogen Corp., Carlsbad, CA) in the presence of a C-6 amine modified random hexamer and aminoallodeoxyuridine to produce fluorescent labeled cDNA. RNA from the reference preparation was all labeled together. Following reverse transcription, RNA was degraded with 0.1 M NaOH and neutralized with ph 7.0 Hepes buffer. Following isolation on Qiaquick columns (Qiagen), elution and lyophilization, the lung-sample was labeled with Cy5 ester and reference cDNA samples with Cy3 ester (Amersham Biotech, Piscataway, NJ) After separation from unbound dye, the samples were again lyophilized and then reconstituted in hybridization solution (32% formamide, 5 X Denhardt's solutions, 3X SSC, 20 mM Hepes buffer, ph 7.0) for 20 hrs at 42 degree. Following hybridization on a microarray (one per rat) and washing to remove unhybridized cDNA and scanning with an Axon 4000B (Axon Instruments, Union City, CA) at 10 micron resolution, the resulting 16-bit TIFF images were analyzed with GenePix 4.1 software (Axon Instruments) for calculation of Cy5 and Cy3 fluorescence intensities at each spot.
Keywords: time-course

Contributor(s)

Bowman PD, Sondeen JL, Zhao B, Dubick MA, Vaughan GM

Citation(s)

16159910

Submission date

Feb 09, 2004

Last update date

Mar 02, 2012

Contact name

Phillip Bowman

E-mail(s)

phillip.bowman@cen.amedd.army.mil