|
| Status |
Public on Jan 30, 2023 |
| Title |
Hdac1 vs WT replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Oregon-R Wild type
|
| Organism |
Drosophila melanogaster |
| Characteristics |
background strain: Oregon-R
genotype/variation: Wild type
Stage: 3rd instar larvae
|
| Growth protocol |
Fly grew at 25 degree C, harvest at 3rd instar stage
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
After DNaseI treatment, RNA were purified by RNeasy kit (Qiagene)
|
| Label |
Alexa Fluor 555
|
| Label protocol |
15 µg of total RNA were primed with 2 µl of 2.5 µg/µl anchored Oligo-(dT)20 primer at 70°C for 10 min, then reversed transcribed at 46°C for 2 h in the presence of 400 U SuperScript III RTase, and 10mM dNTP including amino-allyl-dUTP. (SuperScript Indirect cDNA Labeling System, Invitrogen). After reaction, add 10μL 0.5M EDTA and 10μL 1N NaOH, incubate at 70℃ for 15 min. to degrade RNA. Neutralize the hydrolysis reaction by adding 25μL 1M HEPES pH 7.4. After clean by QIAquick(Qaigen), cDNA were elute from column by 60 μl 0.1M NaHCO3 pH 9.0. Add elute into a tube contain Alexa 555 or 647 NHS ester. Couple the dye at room temperature in the dark for 60-90 min. Stop the reaction by adding 15 μl of 4 M NH2OH-HCl in the dark for 15 min. Immediately purify the labeled cDNA through QIAquick column.
|
| |
|
| Channel 2 |
| Source name |
Oregon-R Hdac1 mutant
|
| Organism |
Drosophila melanogaster |
| Characteristics |
background strain: Oregon-R
genotype/variation: Hdac1[303]/Hdac1[def8]
Stage: 3rd instar larvae
|
| Growth protocol |
Fly grew at 25 degree C, harvest at 3rd instar stage
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
After DNaseI treatment, RNA were purified by RNeasy kit (Qiagene)
|
| Label |
Alexa Fluor 647
|
| Label protocol |
15 µg of total RNA were primed with 2 µl of 2.5 µg/µl anchored Oligo-(dT)20 primer at 70°C for 10 min, then reversed transcribed at 46°C for 2 h in the presence of 400 U SuperScript III RTase, and 10mM dNTP including amino-allyl-dUTP. (SuperScript Indirect cDNA Labeling System, Invitrogen). After reaction, add 10μL 0.5M EDTA and 10μL 1N NaOH, incubate at 70℃ for 15 min. to degrade RNA. Neutralize the hydrolysis reaction by adding 25μL 1M HEPES pH 7.4. After clean by QIAquick(Qaigen), cDNA were elute from column by 60 μl 0.1M NaHCO3 pH 9.0. Add elute into a tube contain Alexa 555 or 647 NHS ester. Couple the dye at room temperature in the dark for 60-90 min. Stop the reaction by adding 15 μl of 4 M NH2OH-HCl in the dark for 15 min. Immediately purify the labeled cDNA through QIAquick column.
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent Hi-RPM Gene Expression Hybridization Kit,) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially by Gene Expression Wash Buffer Kit and dry.
|
| Scan protocol |
Scanned on an Agilent G2565CA scanner.
Images were quantified using Agilent Feature Extraction Software (version A.10.5.1.1).
|
| Description |
Biological replicate 3 of 4. WT or Hdac1 mutant were harvest at 3rd instar stage
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Oct 17, 2012 |
| Last update date |
Jan 30, 2023 |
| Contact name |
Der-Hwa Huang |
| E-mail(s) |
mbdhuang@ccvax.sinica.edu.tw
|
| Organization name |
Academia Sinica
|
| Department |
Insititute of Molecular Biology
|
| Lab |
N420
|
| Street address |
128, Sec2, Academia Rd.
|
| City |
Nankang, Taipei |
| State/province |
Taiwan |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL16193 |
| Series (1) |
| GSE41640 |
Oregon-R: Control vs. Hdac1 mutant |
|
