|
| Status |
Public on Dec 05, 2009 |
| Title |
Rh3 - CD34+ GCSF plus AMD3100 - mAdbID:98307 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pooled Normal Donor PBMC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
cell type: PBMC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction with 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 Labeling Protocol
Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy3) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
|
| |
|
| Channel 2 |
| Source name |
CD34+ GCSF plus AMD3100 Mobilized
|
| Organism |
Macaca mulatta |
| Characteristics |
tissue: blood
cell type: CD34+
|
| Treatment protocol |
Treatment type: compound
Agent: GCSF plus AMD3100
Treatment dose: 10 ug / kg (GCSF); 1mg / kg (AMD3100)
Treatment time: 5 days (GCSF); 2 hours (AMD3100)
In-vivo treatment: Rhesus macaques recieved GCSF for 5 days. On the 5th day donors recieved one dose of AMD3100. Then the PBSC were collected by aphoresis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction with 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 Labeling Protocol
Other: Target labeling by reverse transcription: 4ul First strand buffer 1ul dN6 primer (8ug/ul; Boehringer Mannheim Cat# 1034731 resuspended in 250ul DEPC H2O) 2ul 10X low T - dNTP (5mM A, C and GTP, 2mM dTTP) 2ul Cy-dUTP (1mM Cy5) 2ul 0.1 M DTT 1ul RNasin 3-6ug amplified mRNA in 7 ul DEPC H2O Mix well and heat to 65C for 5min then cool down to 42C. Add 1.5ul SSII and Incubate for 90 min at 42C. Add 2.5ul 500mM EDTA and heat to 65C for 1min. Add 5ul 1M NaOH and incubate at 65C for 15min to hydrolyze RNA. Add 12.5ul 1M Tris immediately to neutralize the pH. Add 35ul of 1xTE. Target clean up: Prepare Bio-6 column and run target solution through it. Collect flow through and add 200ul 1xTE. Concentrate target to ~20ul using a microcon YM-30 column (Millipore Cat# 42410).
|
| |
|
| |
| Hybridization protocol |
Sample Hybridization Protocol
Other: Hybridization: Combine Cy3 and Cy5 labeled target (adjust the color to purple) and complete dry the sample by speed vacuum . Add 37ul of the following mixture to the dried target (for 20x40mm slide): 1ul 50x Denhardt’s blocking solution (Sigma Cat# 2532) 1ul poly dA (8mg/ml Pharmacia Cat# 27-7988-01) 1ul yeast tRNA (4mg/ml Sigma Cat# R8759) 10ul Human Cot I DNA (1mg/ml Gibco BRL Cat# 15279-011) 3ul 20X SSC 1ul 10% SDS 20ul of DEPC H2O Heat for 2 min at 99oC and Cool to RT. Apply target mixture to array slide, add coverslip, place in humidified hyb chamber, and hybridize at 65oC over night.
|
| Scan protocol |
Creator: GenePix Pro 4.0.1.17
Scanner: GenePix 4000B [84945]
ScanPower: 33;; 33
LaserPower: 3.15;; 3.51
Temperature: 30.02
|
| Description |
mAdb experiment ID: 98307
|
| Data processing |
BRB ArrayTool Data Processing
Calculation Method: 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 < 300, and spot size less than 25um 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.
|
| |
|
| Submission date |
Jul 02, 2009 |
| Last update date |
Dec 05, 2009 |
| Contact name |
Francesco Maria Marincola |
| E-mail(s) |
fmarincola@sidra.org
|
| Phone |
301-793-8210
|
| Organization name |
Sidra Medical and Research Center
|
| Street address |
Al Nasr Tower, AL Corniche Street, PO Box 26999
|
| City |
Doha |
| ZIP/Postal code |
PO Box 26999 |
| Country |
Qatar |
| |
|
| Platform ID |
GPL8793 |
| Series (1) |
| GSE16936 |
AMD3100 and G-CSF Mobilize Different CD34+ Cell Populations Based on Gene and miRNA Expression |
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