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Sample GSM1446223 Query DataSets for GSM1446223
Status Public on Jul 26, 2014
Title RNA_human-monocyte-derived-dendritic-cell_Leishmania-major-strain-FV1-lpg1-addback-mutant-infected_8hpi_donor-170
Sample type RNA
Source name human-MDDCs_L. major-strain-FV1-lpg1-addback-mutant-infected_8hr-post-infection
Organism Homo sapiens
Characteristics host cell type: Monocyte derived dendritic cells (MDDCs)
donor id number: 170
infecting parasite species: Leishmania major
parasite strain: FV1
parasite genotype/variation: lpg1 gene knockout add-back
hours post-infection (hpi): 8 hours
background: 240.694
Treatment protocol After 8 hours post-infection, MDDCs were harvested washing in medium and application of 0.5 mM EDTA (Gibco) in 1X PBS (Cellgro). Cell harvests were spun down, washed, and pellets resuspended in RNAlater RNA Stabilization Solution (Applied Biosystems).
Growth protocol CD14+ monocytes of four anonymous healthy adult female human donors (Central Indiana Regional Blood Center, Indianapolis, IN) were isolated from peripheral blood mononuclear cells by positive selection using an AutoMACS separator (Miltenyi Biotec) after isolation using lymphocyte separating media (Cellgro) following manufacturer protocols. The purity of separated monocytes was determined by flow cytometry analysis on a MCL500 flow cytometer (Beckman Coulter) using fluorescent antibody staining for CD14, CD19, CD45, and CD3 (Biolegend). Enriched monocytes were differentiated into dendritic cells (MDDC) by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF; 2,000 U/mL) and interleukin-4 (IL-4; 800 U/mL) recombinant cytokines (Peprotech) on days 0, 3, and 6 after plating in six-well plates at 2.5 × 106 cells/mL in complete RPMI medium (10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine). Immature MDDC were harvested on day 7 and plated in 6-well plates at 2 × 106 cells in 2 mL complete RPMI medium per well in the absence of exogenous cytokine. The phenotype puritiess of DCs was determined by flow cytometry analysis of CD1a, CD14, HLA-DR, CD40, CD80, and CD86 expression. All infections were performed with Leishmania major Friedlin V1 strain (NIH) and gene knockout and add-back mutants for LPG1 [encoding a galactofuranosyl transferase] or LPG2 [encoding a golgi GDP-Man transporter] genes. All parasites were cultured at 26°C without CO2 in complete medium 199 (20% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine in 50 mM HEPES, 5 mg/mL hemin in 50% triethanolamine, and 1 mg/mL biotin). Parasites tested negative for mycoplasma using a Mycoplasma PCR detection set (Takara) and tested below the detection limits for endotoxin using a Limulus Amoebocyte Lysate kit (Charles River Laboratories). Infective-stage metacyclic promastigotes were isolated by use of a Ficoll gradient as previously described (PMID: 11748963) and opsonized with 5% normal human serum prior to infection at a concentration of ten parasites to one host cell. Infection rates were determined at the end of each experiment by DiffQuick staining of cytospin whole cell preparations and visualization by light microscopy.
Extracted molecule total RNA
Extraction protocol Total RNA from uninfected or Leishmania-infected human MDDCs was isolated using an RNEasy kit (Qiagen). Bioanalyzer 2100 (Agilent Technologies) and RNA 6000 Nano kits (Agilent Technologies) were used to determine total RNA integrity.
Label Cy3
Label protocol 25ng of high quality RNA was converted to double stranded cDNA using Sigma TransPlex Complete Whole Transcriptome Amplification Kit (Sigma). RNA degradation, double stranded cDNA purification, and cDNA precipitation was conducted following NimbleGen Gene Expression Array user’s guide protocols (Roche NimbleGen). A Nanodrop ND-2000 (ThermoScientific) was used to determine total RNA and double stranded cDNA concentrations. Sample cDNAs were Cy3-labeled using NimbleGen Single Color Labeling Kit (Roche NimbleGen) per manufacturer's recommendations.
Hybridization protocol Labeled cDNAs were hybridized to 12-plex NimbleGen Homo sapiens Expression Arrays (platform accession number GPL16025), featuring 45,033 probes representing 23,752 gene transcripts, using Hybridization Kit, LS (Roche NimbleGen) and Wash Buffer Kit (Roche NimbleGen) per manufacturer's recommendations.
Scan protocol Image acquisition of arrays was performed using a NimbleGen MS 200 Microarray Scanner, at a 2 micron resolution.
Description This sample is expression values of total mRNA taken from cytokine stimulated human monocyte derived dendritic cells infected with lpg1 gene knockout add-backs Leishmania major (FV1 strain) parasites and is the first of four biological replicates
Data processing NimbleGen array image data were processed using NimbleScan (version 2.5) to extract intensity values for each gene. NimbleScan software automates the pre-processing of NimbleGen microarray image data, including identifying the location of each probe, extraction of intensity data from the image, background correction, and obtaining expression summary values for each gene using a probe-level summarization method RMA (Robust Multi-Array Average).
Values are hybridization fluorescence intensity averages of RMA normalized gene transcript probesets (consisting of three probes per gene transcript). All sample values are reported for any probeset with an intensity value in at least one of the 24 samples which was two times greater than that of the corresponding sample array background. Such values were subsequently used in downstream analysis. All values for any probeset that did not pass this filter are listed as "NULL".
Submission date Jul 25, 2014
Last update date Dec 01, 2014
Contact name Mary Ann McDowell
Organization name University of Notre Dame
Department Biological Sciences
Street address 210 Galvin Life Science Center
City Notre Dame
State/province Indiana
ZIP/Postal code 46556
Country USA
Platform ID GPL16025
Series (1)
GSE59766 Leishmania major Friedlin V1 lpg1- and lpg2- mutants differentially modulate the Interleukin-12 immune response in human dendritic cells.

Data table header descriptions
VALUE RMA normalized

Data table
AB000409 4806.7899
AB000463 1898.4866
AB000781 NULL
AB001328 NULL
AB002294 1141.8004
AB002308 4267.7691
AB002311 2341.6949
AB002313 9963.7094
AB002360 NULL
AB002377 939.8
AB002381 1129.7849
AB002382 2362.9395
AB002384 NULL
AB003177 2032.4289
AB003333 11830.4212
AB006589 NULL
AB006590 NULL
AB006621 NULL
AB006625 NULL
AB007457 7357.6634

Total number of rows: 45033

Table truncated, full table size 754 Kbytes.

Supplementary file Size Download File type/resource
GSM1446223_563422_2014-03-27_Cycle4_532_170LPG1AB.pair.gz 2.9 Mb (ftp)(http) PAIR
Raw data provided as supplementary file
Processed data included within Sample table

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