Strain: tud Progeny Gender: Female Age: 72 hr APF Tissue: Whole Body
Treatment protocol
Female tud progeny were collected at the white pre-pupal stage between Zeitgeber time (ZT) 1 and ZT 4, aged at 25◦C for 72 hours and then snap frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from ~30 pupae by homogenization and extraction using the TRIzol® protocol (Invitrogen, Carlsbad, CA) and resuspended in 20µL diethylpyrocarbonate (DEPC)-treated H2O.
Label
Cy5
Label protocol
cDNAs were directly labeled with Cy5 or Cy3 during the reverse transcription reaction; 30µg of total RNA was used as a starting template. The reverse transcription reaction was performed for two hours at 42◦C using the following reagents (values in parentheses are final molarity or final concentration): oligo dT primer (Operon, 3.75 μM), dithiothreitol (Invitrogen, 10 mM), First Strand Buffer (Invitrogen, 1×), dNTPs minus dTTP (Invitrogen, 0.5 mM), dTTP (Invitrogen, 50 μM), Cy-labeled dUTP (Perkin-Elmer, 0.625 nM), and Superscript II reverse transcriptase (Invitrogen, 10 U/μL). The reaction was stopped and RNA was hydrolyzed by a 20 minute incubation at 65◦C with NaOH (167 mM) and EDTA (83 mM). After neutralizing by adding HEPES buffer (pH8.0; 294 mM) and sodium acetate (pH5.2; 228 mM), cDNA samples were purified (Gel-Purification Kit, Qiagen, Valencia, CA).
Strain: Canton S Gender: Male and Female Age: All pupal stages Tissue: Whole Body
Treatment protocol
Male and female flies of all pupal stages were collected and snap frozen in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from the entire batch by homogenization and extraction using the TRIzol(R) protocol (Invitrogen, Carlsbad, CA) and 30 ug were resuspended in 20µL diethylpyrocarbonate (DEPC)-treated H2O.
Label
Cy3
Label protocol
cDNAs were directly labeled with Cy5 or Cy3 during the reverse transcription reaction; 30µg of total RNA was used as a starting template. The reverse transcription reaction was performed for two hours at 42◦C using the following reagents (values in parentheses are final molarity or final concentration): oligo dT primer (Operon, 3.75 μM), dithiothreitol (Invitrogen, 10 mM), First Strand Buffer (Invitrogen, 1×), dNTPs minus dTTP (Invitrogen, 0.5 mM), dTTP (Invitrogen, 50 μM), Cy-labeled dUTP (Perkin-Elmer, 0.625 nM), and Superscript II reverse transcriptase (Invitrogen, 10 U/μL). The reaction was stopped and RNA was hydrolyzed by a 20 minute incubation at 65◦C with NaOH (167 mM) and EDTA (83 mM). After neutralizing by adding HEPES buffer (pH8.0; 294 mM) and sodium acetate (pH5.2; 228 mM), cDNA samples were purified (Gel-Purification Kit, Qiagen, Valencia, CA).
Hybridization protocol
cDNA samples were then dried and resuspended in formamide (56%), sodium citrate buffer (SSC, 3.37×), SDS (1.12%), Denhardts (5.62×), and Polyadenylic acid potassium salt (0.9 mg/ml; Sigma-Aldrich, St. Louis, MO). Samples were boiled for 2 minutes and then applied to microarray slides underneath a LifterSlip (Erie Scientific, Portsmouth, PA). Microarrays were hybridized at 42◦C for 14-18 hours, then washed in a solution of 1.5% SDS and 1X SSC for 5 minutes, a solution of 0.20X SSC for 5 minutes, and two solutions of 0.05X SSC for 10 minutes each.
Scan protocol
All arrays were scanned using the GenePix 4100A scanner and GenePix Pro 5.0 software from Axon Instruments (Molecular Diagnostics, Sunnyvale, CA).
Description
None
Data processing
Visual inspection of the microarray images filtered out florescence most likely not due to labeled cDNA binding; the data from these array elements was discarded. Array elements were only considered for further analysis if at least one channel (Cy3 or Cy5) had greater than 75% of the pixels with intensity values one standard deviation above background levels. All microarray normalization and statistical analyses were performed using the limma package of BioConductor in the program R.