|
| Status |
Public on Jul 16, 2010 |
| Title |
Resistant versus Sensitive replicate 3 dye swap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NP-18AR3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NP-18AR3 pancreatic cancer derived cell line
sensitivity: TK/GCV resistant cell line 3
source: biological replicate 3, cultured cells from independently generated clone, different from NP-18AR1 and NP-18AR2
|
| Growth protocol |
NP-18 and NP-18AR cells were cultured in RPMI medium supplemented with 10% FBS and penicillin (10.000U/ml) and streptomycin (10 mg/ml), at 37ºC and 5% CO2 and 100% humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a commercial kit (Rneasy, Qiagen)
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).
|
| |
|
| Channel 2 |
| Source name |
NP-18
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NP-18 pancreatic cancer derived cell line
sensitivity: TK/GCV sensitive cell line
source: biological replicate 3, third of three independently treated cell cultures from same parental clone NP-18
|
| Growth protocol |
NP-18 and NP-18AR cells were cultured in RPMI medium supplemented with 10% FBS and penicillin (10.000U/ml) and streptomycin (10 mg/ml), at 37ºC and 5% CO2 and 100% humidity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a commercial kit (Rneasy, Qiagen)
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA were reverse transcribed, amplified, labelled by in vitro transcription using the Low Input Linear Amplification Kit (Agilent 5184-3523), and hybridized following the manufacturer’s instructions (SSC wash Agilent 60-mer oligo microarray processing protocol, Version 4.1, April 2004).
|
| |
|
| |
| Hybridization protocol |
Three biological replicate experiments were performed confronting TK/GCV resistant cell lines versus de parental TK/GCV sensitive NP-18 pancreatic cancer derived cell line. For all three three biological replicates each experimental pair of labelled samples was co-hybridized on at least two separate microarrays with dye swapping to correct for dye bias effects. Thus, up to nine microarray hybridizations were processed , totalling 9 array datasets.
|
| Scan protocol |
Fluorescent images were obtained using an Agilent G2565BA scanner at 100% PMT 100% laser power settings and quantified through the GenePix 6.0 software (Axon, Molecular Devices, Sunnywale, CA) using the irregular feature finding option.
|
| Description |
NP-18AR versus NP-18 biological replicate 3 of 3. Technical replicate 2 of 3.
|
| Data processing |
Extracted raw data were filtered and normalized using the Limma package developed within the Bioconductor project in the R statistical programming environment. The two channels were balanced by lowess normalization using 0.3 as the span parameter with reduced weights in control and poor quality spots, followed by scaling across chips. Target genes were considered as differentially expressed when above the 95% rank in the B empirical Bayes statistic and a set fold change. All quantitative and statistical analyses were performed using using Limma package in the R. environment (Smyth 2004).
|
| |
|
| Submission date |
Jul 16, 2009 |
| Last update date |
Jul 16, 2009 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL887 |
| Series (1) |
| GSE17137 |
NP-18 vs. NP-18AR transcriptional profile upon treatment with TK/GCV |
|
