|
| Status |
Public on Feb 04, 2014 |
| Title |
T47D_expression_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
breast cancer cell T47D-MTVL (breast cancer cell line carrying one stably integrated copy of luciferase reporter gene driven by the MMTV promoter)
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
tissue: breast cancer ductal carcinoma
cell line: T47D-MTVL
|
| Growth protocol |
T47D-MTVL (RPMI 1640 medium + 10%FBS, 1% glutamine, 1% penicillin/streptomycin)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using High Pure RNA isolation kit (Roche) according to the manufacturer's instructions. cDNA was obtained from 100 ng of total RNA using SuperScript VILO cDNA synthesis (Invitrogen). High RNA integrity was assessed by Bioanalyzer nano 6000 assay with RIN numbers 9.5 fopr T47D_r1 and 9.2 for T47D_r2
|
| Label |
Cy3
|
| Label protocol |
For each sample 100 ng of total RNA mixed with 2 ul of 1:10000 Agilent One Color Spike Mix were reversed transcribed into cDNA with a T7 promoter and the cDNA was in vitro transcribed into cRNA in the presence of Cy3-CTP using the Low input quick Amp kit (Agilent). Labelled samples were purified using RNeasy mini spin columns (Qagen) and yield and specific activity wwere assessed by spectrophotometry (Nanodrop) and confirmed to be within specifications (>825 ng and >6 pmol Cy3/ug cRNA). 600 ng of cRNA were preblocked and fragmented in Agilent fragmentation buffer and mixed with Agilent GEx Hybridization mix.
|
| |
|
| Hybridization protocol |
Hybridization mix was laid onto each sector of subarray gasket slide and sandwiched against a 8 x 65K format oligonucleotide microarray (Human v1 Sureprint G3 Human GE 8x60k Microarray, Agilent design ID 028004) inside a hybridization chamber which wqs hybridized overnight at 65ºC in a rotisserie oven rotating at 10 rpm for a total of 17 hours. Subsequently array chambers were disassembled submerged in Agilent Gene Expression Buffer 1 and washed 1 minute in another dish with the same solution with a magnetic stirrer at 200 rpm at room temperature, followed by 1 minute in gilent Gene Expression Buffer 2 with a magnetic stirrer at 200 rpm at 37ºC and immediate withdrawal from the solution and air drying.
|
| Scan protocol |
Fluorescent signal was captured into TIF images with an Agilent scanner using recommended settings (20 bit, 5 um resolution, 100% PMT and 100% laser power) with Scan Control software (Agilent).
|
| Description |
T47D-MTVL cell line under basal growth conditions, biological replicate 2
|
| Data processing |
Signal intensities were extracted into a tabulated text file using Feature Extraction software (Agilent) using the appropriate array configuration and annotation files. The normalized log2intensities were obtained using quantile method with normexp background correction the Bioconductor Limma package in R.
|
| |
|
| Submission date |
Jul 25, 2013 |
| Last update date |
Feb 04, 2014 |
| Contact name |
Albert Jordan |
| E-mail(s) |
ajvbmc@ibmb.csic.es
|
| Organization name |
IBMB-CSIC
|
| Department |
Molecular Genomics
|
| Street address |
Baldiri Reixac 4
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (2) |
| GSE49236 |
Gene expression of T47D-MTVL human breast cancer cells growing in normal conditions |
| GSE49345 |
Genomic distribution of human histone H1 subtypes |
|
