Minimum Information About a Microarray Experiment
MIAME – Checklist
A Network Based Analysis of Systematic Inflammation in Humans
S. Calvano and Wenzhong Xiao, et. al.
Experiment
Experimental design:
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Type of experiment: Gene expression profiling of human blood
leukocytes in response to in vivo endotoxin administration.
Experimental factors: Healthy male and female subjects were intravenously
administered either endotoxin or only vehicle (control). Arterial blood samples
were collected before infusion (0 hours) and at post infusion times of 2, 4, 6,
9, and 24 hours. Blood leukocytes were isolated and analyzed using
Affymetrix GeneChipTM arrays
Number of hybridizations: 48 Human U133A and 48 U133B oligonucleotide
arrays (Affymetrix)
Quality Controls: All arrays passed quality control by dChip. Probe sets,
whose signal intensity was identified as being absent by Microarray Suite v5
(Affymetrix) on all arrays, were not included in further analysis.
Supplemental website: http://web8.mgh.harvard.edu/review/InflammationAnalysis/index.htm under construction; see also http://www.gluegrant.org
Samples used, extract preparation and labeling:
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The origin of the biological sample: Human experimental endotoxicosis.
Eight healthy male and female subjects between 18 and 40 years of age were
intravenously administered either NIH Clinical Center Reference Endotoxin,
(CC-RE-Lot 2) at a dose of 2 ng/kg BW or 0.9% sodium chloride vehicle over
a 5 minute period. Arterial blood samples were collected before endotoxin
infusion (0 hours) and at post infusion times of 2,4,6,9, and 24 hours.
Manipulation of biological samples and protocols used: 1. Blood
Sampling. Blood was collected and bicarbonate-buffered ammonium
chloride solution (0.826% NH4Cl, 0.1% KHC03, 0.0037% Na4EDTA in H2O)
was added at a ratio of 20:1 (lysis buffer: blood). The samples were
subsequently incubated at room temperature until erythrocyte lysis was
complete (approximately 5 to 7 minutes). The leukocytes were then
recovered by centrifugation (400 x g, 4º C) and washed once in ice-cold
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phosphate buffered saline. The leukocyte pellets were then resuspended in 8
ml of RLT buffer (Qiagen Inc., Valencia, CA) and the samples sheared ten
times with an 18 gauge needle attached to a 10 ml syringe. Samples were
then immediately frozen at -70º C until the RNA was extracted. 2. Leukocyte
RNA Isolation. Total cellular RNA was isolated from the leukocyte pellets
using a commercial kit (RNeasy, Qiagen, Inc); purity was confirmed by
spectrophotometry (A260/A280 ratio), and capillary electrophoresis (Agilent
2100 Bioanalyzer, Agilent Inc., Palo Alto, CA) .
Labelling protocol: Complementary RNA (cRNA) synthesis. cRNA
synthesis was performed with 4 µg of total cellular RNA, and labelled based
on the protocol outlined by Affymetrix Inc. (Santa Clara, CA), with few
modifications.
Hybridization procedures and parameters:
The protocol and conditions used during hybridization, blocking and washing are
provided in the Expression Analysis Technical Manual by Affymetrix Inc. (Santa
Clara, CA), and available at
http://www.affymetrix.com/Auth/support/downloads/manuals/expression_print_m
anual.zip .
Measurement data and specifications:
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GeneChipTM arrays were scanned using the HP GeneArray Scanner
(Affymetrix). The .cel files were generated using the Microarray Suite v5
software from Affymetrix
(http://www.affymetrix.com/Auth/support/downloads/manuals/mas_manual
.zip).
44,924 probe sets on the U133A and U133B arrays were analyzed.
Normalization was preformed using dChip (http://www.dchip.org/), and
expression level was modeled using the perfect match only model. Probe
sets, whose signal intensity was identified as being absent by Microarray
Suite v5 (Affymetrix) on all arrays, were not included in further analysis.
Array Design
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General array design: in situ synthesized arrays by Affymetrix, and
commercially available from Affymetrix.
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The arrays used in this study can be purchased from Affymetrix:
http://www.affymetrix.com/products/arrays/specific/hgu133.affx, P/N#
90044