E-GEOD-135 - Transcription profiling of mouse erythroid progenitor (EPC) derived from splenocytes at stage I, II and III
Submitted on 26 December 2002, released on 27 October 2007, last updated on 10 June 2011
Expression profiles were analyzed (via Affymetrix MG-U74Av2 arrays) at three distinct stages of late erythroid progenitor cell development. Stages correspond to Kit(+)/EpoR-high/CD71(+)/Ter119(-) proerythroblasts [stage I]; Kit-low/EpoR(+)/CD71-high/Ter119(+) polychromatophilic erythroblasts [stage II]; and Kit(-)/EpoR-low/CD71-low/Ter119-high/lowFALS normoblasts [stage III]. Primary splenic (pro)erythroblasts were prepared at 80, 100 and 120 hours post thiamphenicol dosing, and were purified by MACS using a transgenic erythroid-restricted “EE” tag. For each stage, duplicate hybridizations were performed. GSM3371 and GSM3372 are results from stage I hybridizations; GSM3373 and GSM3374 are results for stage II; and GSM3375 and GSM3376 are results for stage III. The order of listed genes is organized based first on average p-value among all six hybridizations in ascending order, and second on fold-increases in expression levels from stage I to stage II (descending order). B] METHOD DETAILS (as a supplement to Zhang et al., submitted) B1-B6 below:; B.1/ ERYTHROID PROGENITOR CELL PREPARATIONS: Thiamphenicol (TAP) (Sigma, St. Louis, MO) was administered on day 1 to 8-week old Gata1-“EE” mice  (15 mg per gram weight in 0.4 mL of water) as a subcutaneous implant in Spectra Por 2 tubing (Spectrum, Houston, TX) . On days 2 through 4, mice were phlebotomized (80mL blood per day), and TAP implants were removed on day 6. Splenocytes were prepared at 80, 100, or 120 hours post TAP withdrawal by disruption of spleens in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2% fetal bovine serum (FBS) and 0.1 U/mL Epo. Cells were passed through a 70 um strainer (Falcon, Franklin Lakes, NJ), collected at 300 x g for 10 minutes, resuspended in 1 mL of phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.2 mM KH2PO4, pH 7.4), exposed for 2 minutes to 9 mL of 0.8% NH4Cl, 0.01 mM Na2EDTA buffered at pH 7.5 with KHCO3. Cells then were collected through 50% FBS in PBS, and washed in DMEM. B.2/ MACS: Splenocytes (3x10^8 cells per 3 spleens per preparation) in 3 mL of 5mM Na2EDTA, 0.5 % BSA in PBS, pH 7.2 (PBE) were incubated for 5 minutes at 15°C with murine IgG Fc fragment (5 μg per mL) (Pierce, Rockford, IL), and pre-absorbed for 20 minutes at 15°C with anti-mouse IgG2a+2b magnetic microbeads (100 μL per mL) (Miltenyi, Bergisch Gladbach, Germany). Samples were passed through a magnetized MACS-LS column (non-specific adsorption) and unbound cells were collected, washed and resuspended (at 1 x 10^8 cells per mL) in 3 mL of PBE buffer. Cells then were incubated at 15°C for 15 minutes with monoclonal antibody EGFR.1 (5 μg per mL) (PharMingen, San Diego, CA), washed in 20 mL of 2°C PBE buffer, resuspended in PBE at 1 x 10^8 cells per mL, and incubated for 20 minutes at 15°C with anti-mouse IgG2a+2b magnetic microbeads (200 μL per mL). Bead-tagged (pro)erythroblasts were washed in 2°C PBE buffer, resuspended in 3 mL PBE, and applied to a MACS-LS column. Columns were washed six times with 3 mL of PBE, and EE-positive cells were recovered in 5 mL of PBE buffer. Overall, 6 x 10^6 stage I, II and III (pro)erythroblasts were purified from 12, 4, and 3 mice, respectively. B.3/ RNA ISOLATION AND ANALYSIS: Purified (pro)erythroblasts (6 x 10^6 cells) were lysed in 1 mL Trizol reagent (Life Technologies, Gaithersburg, MD). Chloroform (0.2 mL) was added and samples were vortexed and microcentrifuged at 7,500 rpm for 15 minutes at 4°C. The aqueous phase (approximately 0.5 mL) was recovered, and extracted using 0.5 mL of Trizol LS reagent plus 0.1 mL of chloroform. From the recovered epi-phase (750 μL), RNA was precipitated using isopropanol (750 μL), collected (10 minutes at 4°C, 12,000 rpm), washed with 80% ethanol, air-dried, and dissolved in 35 μl of 70°C DEPC-treated water. In Northern blotting, RNA samples were electrophoresed in 6% formaldehyde 1.2% agarose gels, blotted to Nytran membranes (Schleicher and Schuell, Keene, NH), and fixed (312 nm radiation for 3 minutes; 2 hours at 80°C under vacuum). In hybridizations, Redivue deoxyadenosine 5'-(α-32P)-triphosphate labeled probes were prepared by random priming (Prime-a-Gene System, Promega, Madison, WI) using 25 ng of the following cDNA fragments: murine Epo receptor, 880 bp Bgl II to Xba I fragment of pUC19ER-DpI3K ; murine beta-major globin, 1,100 bp Bgl II to Xba I fragment of pGEM7-betamaj-globin ; murine Kit, 1,500 bp BamH I to Nhe I fragment of pRep4DEB-cKit ; murine Gata-1, Xho I fragment of pBluescript-GATA-1 ; 32P-labeled probes were isolated using Sephadex G-50 microcolumns (Pharmacia Biotech, Piscataway, NJ). Hybridizations were for 4 hours at 68°C using 2 × 10^6 cpm of probe per mL in QuickHyb solution (Stratagene, La Jolla, CA). Membranes were exposed to X-OMAT AR film (Kodak, Rochester, NY) and were assayed quantitatively by phosphor imaging (Storm 860 system, Molecular Dynamics, Sunnyvale, CA). B.4/ BIOTIN-cRNA PREPARATION, AND ARRAY HYBRIDIZATIONS: First-strand cDNA was synthesized with a T7-oligo (dT) 24 primer (5'-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-(dT) 24-3') (Geneset, La Jolla CA) and Superscript II reverse transcriptase (Invitrogen, Carsbad, CA) (1 hour, 42°C). DNA polymerase I (cat# 18010-017, Invitrogen) and RNase H (cat# 18021-041, Invitrogen) were used in second strand syntheses (2 hours, 16°C). Products were extracted in Tris-phenol: chloroform: isoamyl (25:24:1) using a phase-lock gel (cat# 175850, Eppendorf 5-prime, Boulder, CO). Precipitation was with 0.5 volumes of 7.5M NH4Ac, 5 μg of glycogen and 2.5 volumes of -20°C absolute ethanol. Double stranded -cDNA (1 μg in 10 µl DEPC-treated water) was used per T7 RNA pol transcription (Enzo BioArray High Yield biotin-UTP and -CTP RNA Labeling Kit) (cat# 42655, Farmingdale, NY). Products were purified using QIAGEN RNeasy columns (cat# 72104) (Valencia, CA). For each sample (16 μg total RNA) approximately 60 μg of purified biotin-labeled cRNA was recovered. Labeled cRNA was fragmented for 35 min at 94°C in 500 mM potassium acetate (C2H3KO2), 150 mM magnesium acetate (C4H6MgO4), 200 mM Tris-acetate (C4H11NO3 • CH3COOH), pH 8.1. Each sample (diluted 1:10 in hybridization buffer) was first hybridized to a Test2 Genechip to confirm full-length transcript representation. Replicate hybridizations were to Affymetrix murine U74Av2 arrays, and data were analyzed using Affymetrix Genechip 5.1 software. B.5/ ANALYSES OF PROFILING OUTCOMES: Transcripts expressed (detected) at each stage were identified based on a detection p-value of <0.05 in one replicate, and <0.1 in the other. 3'/5' ratios of housekeeping genes confirmed uniform biotin cRNA syntheses. Pair-wise comparisons among stages included 4659 transcripts detected in at least one stage, and employed averages of log expression values from duplicate hybridizations for each gene. In these comparisons, modulation is defined by vertical distances from a regression (no-modulation) trendline. For the above 4659 detected transcripts, mean expression values were normalized to account for chip/sample variability by subtracting the stage mean, and dividing by the stage standard deviation. The three normalized values of each gene (one for each stage) then were standardized by subtracting the gene mean and dividing by the gene standard deviation. Non-modulated genes (less than two-fold change) then were removed from further consideration. Clustering of the resulting data was performed by K-means (S+ package), a partitioning algorithm that pursues a minimum of the within-clusters sums of squares (WSS, which measures square Euclidean distances between cluster points and the cluster average within each cluster) . Principal component analysis (PCA) was used to visualize K-means clustering results. PCA is a common method for reduction of dimensionality.
transcription profiling by array, co-expression, time series