|
| Status |
Public on Mar 12, 2014 |
| Title |
SLE_treated_patient5_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Monocytes from SLE patients after fluvastatin treatment
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Monocytes
gender: Female
age: 32
|
| Treatment protocol |
Twenty seven SLE patients were given 20mg/day fluvastatin for one month. Blood samples were obtained before the starting of the treatment and at the end of treatment. Monocytes were isolated by immunomagnetic depletion of non-monocytes.Complementary RNAs from monocyte-RNA isolated of six SLE patients (four before treatment and two after treatment) were prepared for hybridization in an Agilent G4112F platform (Whole human Genome microarray 44K) using the One-color gene expression system (Agilent technologies).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from monocytes was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 500ng RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1,5 ug of Cy3-labelled cRNA (specific activity >10,0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 25 x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55ul of 2x Hi-RPM Agilent hybridization buffer was added to the fragmentation mixture and hybridized to 4x44k Agilent Whole Human Genome Oligo Microarrays (AMADID 14850) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were dissasembled in GE Wash buffer 1 (Agilent) and washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash Buffer 2 (Agilent), then dried immediatly by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 4x44 array slides (Scan Area 61x21,6mm, Scan resolution 5 um, Dye channel is set to Green and Green PMI gain is set to 100%).
|
| Description |
Gene expression after treatment with Fluvastatin
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10,1 (Agilent) using default parameters (protocol GE1-v1_101 and Grid: 014850_D_F_20120130) to obtain bacground subtracted and spatially detrended Processes Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Mar 22, 2013 |
| Last update date |
Mar 12, 2014 |
| Contact name |
Rosario Lopez Pedrera |
| E-mail(s) |
[email protected]
|
| Organization name |
IMIBIC
|
| Street address |
Avda. Menendez Pidal s/n
|
| City |
Cordoba |
| ZIP/Postal code |
14004 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE45422 |
Effects of fluvastatin treatment on monocyte gene expression in SLE patients |
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