Treatment type: compound Agent: AMD3100 Treatment dose: 240ug / kg (AMD3100) Treatment time: 12 hours Other: Healthy donors recieved one dose of AMD3100. Then the PBSC were collected by aphoresis.
Extracted molecule
total RNA
Extraction protocol
RNA extraction with TRIZOL Extraction method: TRIZOL Other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturers instructions. RNA integrity was assessed by the presence of 28S and 18S bands using Agilent bioanalyzer and quantified using nanodrop with A260/A280 ratios > 2.0 after dilution in DEPC water.
Label
cy5
Label protocol
Cy5 Sample Labeling Protocol Other: Small RNA was enriched from 10ug total RNA by flashPAGE (Pre-cast Gel,Type A) (Ambion, Austin, TX, USA) and purified using flashPAGE reaction clean-up kit (Ambion, Austin, TX, USA) according to manufacture’s instruction. The same procedures were applied to obtain small RNA from the Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines that was used as the reference for the miRNA expression array assay. Fragmented small RNA were 3’-end tailed with amine-modified nucleotides and chemically coupled to CyDye fluors (Amersham Biosciences, piscatway, NJ, USA), which sample with Cy5 and the reference with Cy3 respectively, using the mirVana miRNA Labeling Kit (Ambion) following the recommended protocol by the manufactures’ instruction. After labeling, the samples and the reference were co-hybridized to the miRNA array at room temperature over night. Both the processed cDNA and the miRNA array slides were scanned by GenePix scanner Pro 4.0 (Axon, Sunnyvale, CA, USA).
RNA extraction with TRIZOL Extraction method: TRIZOL Other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturers instructions. RNA integrity was assessed by the presence of 28S and 18S bands using Agilent bioanalyzer and quantified using nanodrop with A260/A280 ratios > 2.0 after dilution in DEPC water.
Label
cy3
Label protocol
Cy3 Sample Labeling Protocol Other: Small RNA was enriched from 10ug total RNA by flashPAGE (Pre-cast Gel,Type A) (Ambion, Austin, TX, USA) and purified using flashPAGE reaction clean-up kit (Ambion, Austin, TX, USA) according to manufacture~Rs instruction. The same procedures were applied to obtain small RNA from the Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines that was used as the reference for the miRNA expression array assay. Fragmented small RNA were 3~R-end tailed with amine-modified nucleotides and chemically coupled to CyDye fluors (Amersham Biosciences, piscatway, NJ, USA), which sample with Cy5 and the reference with Cy3 respectively, using the mirVana miRNA Labeling Kit (Ambion) following the recommended protocol by the manufactures~R instruction. After labeling, the samples and the reference were co-hybridized to the miRNA array at room temperature over night. Both the processed cDNA and the miRNA array slides were scanned by GenePix scanner Pro 4.0 (Axon, Sunnyvale, CA, USA).
Hybridization protocol
Sample Hybridization Protocol Other: Small RNA was enriched from 10ug total RNA by flashPAGE (Pre-cast Gel,Type A) (Ambion, Austin, TX, USA) and purified using flashPAGE reaction clean-up kit (Ambion, Austin, TX, USA) according to manufacture’s instruction. The same procedures were applied to obtain small RNA from the Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines that was used as the reference for the miRNA expression array assay. Fragmented small RNA were 3’-end tailed with amine-modified nucleotides and chemically coupled to CyDye fluors (Amersham Biosciences, piscatway, NJ, USA), which sample with Cy5 and the reference with Cy3 respectively, using the mirVana miRNA Labeling Kit (Ambion) following the recommended protocol by the manufactures’ instruction. After labeling, the samples and the reference were co-hybridized to the miRNA array at room temperature over night. Both the processed cDNA and the miRNA array slides were scanned by GenePix scanner Pro 4.0 (Axon, Sunnyvale, CA, USA).
BRBArrayTools Calculation Method: Fluorescence intensities generated by Cy5 and Cy3 probes hybridized onto the microarray slides were scanned were scanned on a GenePix 4000 (Axon Instruments, Union City, CA) at variable PMT voltage to obtain maximal signal intensities with < 1% probe saturation. Data were uploaded to National Institute of Health, NCI/CCR µArray database (http://nciarray.nci.nih.gov). Raw data were retrieved in BRB-ArrayTools(http://linus.nci.nih.gov/BRBArrayTools.html) format for analysis and normalization. Following filter criteria was applied to the data set: 1. spots were excluded if intensity < 100, and spot size less than 10um 2. genes were filtered out if >80% of experiment with missing data values. 3. log2 transformed data were normalized using lowess smoother, and intensity ratios were truncated if greater than 64. Transcriptional data were process by averaging results in replicate experiments for the same genes. Hierchical clustering analysis was performed on BRBArray tools using centered correlation as similarity metric and centroid linkage method: Cluster 3:0. Heat maps were generated using Eisen’s TreView Software.
