|
| Status |
Public on Dec 29, 2011 |
| Title |
Bone_visceral bone metastasis patient 12 |
| Sample type |
RNA |
| |
|
| Source name |
Bone metastasis from breast cancer patient
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
tissue: bone metastasis biopsy
metastasis: Bone_Visceral
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from bone biopsies of human bone metastases using TRIzol (Invitrogen) procedure following the manufacturer’s instructions, and quantified using the invitrogen Qbit kit RNA high sensitivity and the Invitrogen Qbit spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). cRNA yield were checked with a classical UV-Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent hUMAN 1a Microarrays (G4110B) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent) and then dried with the Agilent Stabilisation and drying solution.
|
| Scan protocol |
Slides were scanned immediately after washing on the AFFYMETRIX 428 DNA Microarray Scanner using one color scan setting for 1x22k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and ).
|
| Data processing |
Jaguar Software1 version 2.0 (Affymetrix) for the acquisition of the microarray image, scanning the cy3 fluorescence. ImaGene1 Software (BioDiscovery, Inc.) to obtain background subtracted and spatially detrended Processed Signal intensities. VectorXpression1 Version 4.1 (Invitrogen) to fully analyze the microarray data.
|
| |
|
| Submission date |
Dec 16, 2011 |
| Last update date |
Dec 29, 2011 |
| Contact name |
Mattia Capulli |
| E-mail(s) |
[email protected]
|
| Organization name |
University of L'Aquila
|
| Street address |
Via Vetoio Coppito 2
|
| City |
L'Aquila |
| ZIP/Postal code |
67100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6087 |
| Series (1) |
| GSE34485 |
Microarray analysis of human bone metastases samples |
|
