|
| Status |
Public on Aug 29, 2007 |
| Title |
Heart WT 42h 48h APF rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT_42hAPF_rep2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Strain : WT (yw; Canton S)
Development temperature : 25°C
Stage : 42hr APF (pupae)
Hand dissected cardiac tubes
Biological replicate n°2
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from 5 dissected cardiac tubes with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected cardiac tubes were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy3.
|
| Label |
Cy3
|
| Label protocol |
20 μg of amino allyl aRNA was labelled according the "Dye Coupling and Labeled aRNA Cleanup" protocol of the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion)
700 pmol of incorporated dye was used for hybridization.
|
| |
|
| Channel 2 |
| Source name |
WT_48hAPF_rep2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Strain : WT (yw; Canton S)
Development temperature : 25°C
Stage : 48hr APF (pupae)
Hand dissected cardiac tubes
Biological replicate n°2
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from 5 dissected cardiac tubes with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected cardiac tubes were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy5.
|
| Label |
Cy5
|
| Label protocol |
20 μg of amino allyl aRNA was labelled according the "Dye Coupling and Labeled aRNA Cleanup" protocol of the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion)
700 pmol of incorporated dye was used for hybridization.
|
| |
|
| |
| Hybridization protocol |
Labeled probes (60 µl) were mixed with 60 µl of 4X hybridization buffer and 120 µl of deionised formamide, denatured at 95°C for 5min and hybridized at 37°C for 12 hours with the Automated Slide Processor (ASP) of Amersham. Slides were successively washed at 37°C in 1X SCC/0.2% SDS for 20 min, in 0.1X SCC/0.2% SDS for 2x10 min, in 0.1X SCC for 10 min, rinsed in isopropanol and air dried.
|
| Scan protocol |
Images were obtained with a GenePix 4200AL (Axon Instruments) at 10 um resolution and 5% of laser power. Fluorescence measurements are made using Array Vision quantification software (Imaging Research Inc).
|
| Description |
Cy3 and Cy5 labelled aRNA samples were mixed in equal proportions (700 pmol) and fragmented with the RNA Fragmentation Reagents (Ambion) to enhance aRNA hybridization, dried up to 60ul of final volume, and hybridized on INDAC oligonucleotide microarrays.
|
| Data processing |
Normalization of primary expression data (from not flagged spots) was performed through two successive steps using both R software packages SMA and LIMMA:
- Lowess normalization to normalize the M-values (Cy3/Cy5) for each array separately (within-array normalization) without prior background correction
- Quantile normalization to the A-values making the density distributions the same across arrays to compare expression intensities between them (between-array normalization).
Final normalized log2 M-values are showed in the VALUE column.
|
| |
|
| Submission date |
May 02, 2007 |
| Last update date |
Aug 29, 2007 |
| Contact name |
Bruno ZEITOUNI |
| E-mail(s) |
zeitouni@ibdml.univ-mrs.fr, perrin@ibdml.univ-mrs.fr
|
| Phone |
+33491269611
|
| Fax |
+33491820682
|
| Organization name |
CNRS
|
| Department |
UMR 6216
|
| Lab |
Institut de Biologie du Développement de Marseille (IBDML)
|
| Street address |
Campus de Luminy Case 907
|
| City |
MARSEILLE Cedex 9 |
| ZIP/Postal code |
13288 |
| Country |
France |
| |
|
| Platform ID |
GPL5112 |
| Series (1) |
| GSE7689 |
Genome-wide expression profiling of Drosophila adult heart organogenesis |
|
