Minimum Information about a Microarray Experiment

MIAME – Checklist

Cell Specific Expression and Pathway Analyses Reveal Alterations in Trauma-Related Human T-Cell and Monocyte Pathways

Laudanski, et. al.

Experiment

Experimental design:

  • Type of experiment: Gene expression profiling of circulating total blood leukocytes, T-Cells, and Monocytes in severe trauma patients and healthy subjects.
  • Experimental factors: Healthy subjects, 7 severely traumatized patients and 7 healthy subjects for transcriptome analysis. Venous blood samples were collected. Total blood leukocytes were isolated, and T-cell and monocyte populations were obtained from two subsequent aliquots of the leukocytes. Total leukocytes, enriched T-cells, and enriched monocytes were analyzed using Affymetrix GeneChipTM arrays
  • Number of hybridizations: 42 Human U133A GeneChipTM arrays (Affymetrix)
  • Quality Controls: All arrays passed quality control by dChip.
  • Supplemental website:

Samples used, extract preparation and labeling:

  • The origin of the biological sample: Human trauma Patients with documented Multiple Organ Failure and Healthy Volunteers.Signed informed consent was obtained from a total of 7 severely traumatized patients and 7 healthy subjects under a protocol approved by the Institutional Review Board (IRB) of the University of Rochester School of Medicine. None of the trauma patients with defined MODS had undiagnosed or untreated infections at the time of blood sampling for microarray analysis. Patients were treated with prophylatic antibiotics as clinically indicated by the trauma service.
  • Manipulation of biological samples and protocols used: 60 mls of patient and 90 mls of healthy subject venous blood were collected and divided into three aliquots. For total leukocyte RNA, one was diluted in 20 volumes of bicarbonate-buffered ammonium chloride solution (0.826% NH4Cl, 0.1% KHC03, 0.0037% Na4EDTA in H2O). The leukocytes were then recovered by centrifugation (400 x g, 4º C) and washed once in ice-cold phosphate buffered saline. The leukocyte pellets were then resuspended in RLT buffer (600 μl/107cells) (Qiagen Inc., Valencia, CA). The samples were sheared by passage through shredder columns (Qiagen) and the eluate was immediately frozen at -70ºC until the RNA was extracted.Enriched T-cell and monocyte populations were obtained from the two subsequent aliquots using a two-step isolation procedure. First, a negative isolation technique feasible for use in clinical settings was employed for initial enrichment of T-cell and monocyte populations, based on the RosetteSep™ technology (StemCell Technologies, VancouverBC). A second purification step was employed based on negative selection using Miltenyi magnetic beads. These protocols for isolating enriched blood T-cells and monocytes are described in the Supplementary Materials section of the manuscript. Isolated T cells and monocytes were vortexed in RLT buffer and processed as described for total leukocytes. The distribution of cell types in the total leukocytes and the enriched T-cell and monocyte preparations were determined by flow cytometry using conjugated antibodies specific for T-cells (CD2+, CD3+); monocytes (C14+, CD33+); neutrophils or granulocytes (CD66b+); B-cells (CD19+) and NK cells (CD56+).
  • Labelling protocol: Complementary RNA (cRNA) synthesis. cRNA synthesis was performed with 2 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 .

Measurement data and specifications:

  • GeneChipTM arrays were scanned using the HP GeneArray Scanner (Affymetrix). The .cel files were generated using the Microarray Suite v5 software from Affymetrix (
  • 22,411 probe sets on the Human U133A arrays were analyzed. Normalization was preformed using dChip ( and expression level was modeled using the perfect match only model.

Array Design

  • General array design: in situ synthesized arrays by Affymetrix, and commercially available from Affymetrix.
  • The arrays used in this study can be purchased from Affymetrix: P/N# 90044