|
| Status |
Public on Apr 01, 2010 |
| Title |
S phase versus G2/M phase replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Sphase 2 hours - 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa
|
| Treatment protocol |
HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and cells were taken after 2 hour release (S phase) and 8 hour release (G2/M phase).
|
| Growth protocol |
HeLa cells were maintained in DMEM high glucose + 10% FCS + penicillin/streptomycin supplement.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using standard total RNA extraction protocol. Then mRNAs were purified with Ambion Poly(A)Purist kit from 20 μg total RNA.
|
| Label |
Cy5
|
| Label protocol |
100 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.
|
| |
|
| Channel 2 |
| Source name |
G2/Mphase 8 hours - 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa
|
| Treatment protocol |
HeLa cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and cells were taken after 2 hour release (S phase) and 8 hour release (G2/M phase).
|
| Growth protocol |
HeLa cells were maintained in DMEM high glucose + 10% FCS + penicillin/streptomycin supplement.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using standard total RNA extraction protocol. Then mRNAs were purified with Ambion Poly(A)Purist kit from 20 μg total RNA.
|
| Label |
Cy3
|
| Label protocol |
100 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.
|
| |
|
| |
| Hybridization protocol |
Three biological replicate experiments were performed to compare the expression of mRNAs between S phase (2 hours release after double thymidine blockade) and G2/M phase (8 hours release after double thymidine blockade). For all three biological replicates each experimental pair of labelled samples was co-hybridized on two separate microarrays with dye swapping to correct for dye bias effects. Thus, in total six microarray hybridizations were processed.
|
| 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 |
S phase versus G2/M phase biological replicate 2 of 3. Technical replicate 1 of 2.
|
| 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 with reduced weights in control and poor quality spots, followed by scaling across chips. Differential expression analysis was performed using empirical Bayes statistics (Limma package, Smyth 2004) and SAM (Significance Analysis of Microarrays, Tusher et al. 2001).
|
| |
|
| Submission date |
Apr 03, 2009 |
| Last update date |
Dec 22, 2009 |
| Contact name |
Isabel Novoa |
| E-mail(s) |
isabel.novoa@crg.es
|
| Organization name |
Center for Genomic Regulation
|
| Department |
Gene Regulation
|
| Street address |
Dr. Aiguader, 88
|
| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL887 |
| Series (1) |
| GSE15547 |
Expression profiling of HeLa cells at S and G2/M phases of mitotic cell cycle. |
|
