|
| Status |
Public on Apr 01, 2010 |
| Title |
ALL fraction mRNAs 2 hours versus SHORT fraction mRNAs 2 hours replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ALL fraction 2h - 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
molecule: ALL fraction RNA
|
| 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 hours release (S phase) and 8 hours release (G2/M phase).
|
| Growth protocol |
HeLa cells were maintained in DMEM high glucose + 10% FCS + penicillin/streptomycin supplement.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using standard total RNA extraction protocol. Then for each condition total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261.
|
| Label |
Cy5
|
| Label protocol |
100 ng of mRNA (ALL fraction mRNA or SHORT fraction mRNA) 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 |
SHORT fraction 2h - 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
molecule: SHORT fraction RNA
|
| 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 hours release (S phase) and 8 hours release (G2/M phase).
|
| Growth protocol |
HeLa cells were maintained in DMEM high glucose + 10% FCS + penicillin/streptomycin supplement.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using standard total RNA extraction protocol. Then for each condition total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261.
|
| Label |
Cy3
|
| Label protocol |
100 ng of mRNA (ALL fraction mRNA or SHORT fraction mRNA) 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 and SHORT fraction mRNAs was used as the common reference to compare with ALL fraction mRNAs. 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, up to six microarray hybridizations were processed for each of the two comparisons - ALL fraction mRNAs versus SHORT fraction mRNAs at S phase (2 hours release after double thymidine blockade) and ALL fraction mRNAs versus SHORT fraction mRNAs at G2/M phase (8 hours release after double thymidine blockade)-, totalling 12 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 |
ALL fraction mRNAs 2 hours versus SHORT fraction mRNAs 2 hours biological replicate 3 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).
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| |
|
| Submission date |
Mar 25, 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) |
| GSE15399 |
Global analysis of differential polyadenylation of mRNAs during mitotic cell cycle: S phase versus G2/M phase. |
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