Investigation Title RNAi knock down of daf-16 in C. elegans over 0-192 hours Comment[Submitted Name] daf-2 RNAi 0-192 hrs Experimental Design time_series_design RNAi profiling by array Experimental Design Term Source REF mo:1.1.3 EFO Comment[ArrayExpressReleaseDate] 2003-07-17 Comment[AEMIAMESCORE] 2 Comment[ArrayExpressAccession] E-MEXP-9 Comment[MAGETAB TimeStamp_Version] 2010-08-11 16:46:39 Last Changed Rev: 13058 Experimental Factor Name Experimental Factor Type Experimental Factor Term Source REF Person Last Name Fraser Kamath Murphy Ahringer McCarroll Bargmann Kenyon Li Person First Name Andrew Ravi Coleen Julie Steven Cornelia Cynthia Hao Person Mid Initials s T a i j Person Email ctmurph@itsa.ucsf.edu Person Phone 415-476-9864 Person Fax 415-514-4145 Person Address 600 16th St. S314 GH Mission Bay, San Francisco, CA, 94143-2200, USA Person Affiliation Wellcome CRC Wellcome CRC Biochemistry and Biophysics, U.C. San Francisco Wellcome CRC UCSF u UCSF UCSF Person Roles submitter Person Roles Term Source REF mo Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2003-07-17 PubMed ID 12845331 Publication DOI 12845331 Publication Author List Coleen Murphy; Steven McCarroll; Cornelia Bargmann; Andrew Fraser; Ravi Kamath; Julie Ahringer; Hao Li; Cynthia Kenyon Publication Title Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans Publication Status journal_article Publication Status Term Source REF mo Experiment Description Comparison of sterile worms grown on Control Vector, daf-2 RNAi, and daf-2 + daf-16 RNAi. Synchronized fer-15(b26); fem-1(hc17) animals were grown on RNAi bacteria at 25ºC, induced with IPTG on Day 1 of adulthood, and collected from 0-48 hours (10 time points) and 0-196 hours (10 time points); RNAi bacteria was supplemented as necessary for later time points. Worms were floated off lawns with M9 buffer, centrifuged, and washed again in M9. The pelleted worms were dissolved in Trizol (Gibco) and frozen in liquid nitrogen.Time courses were compared with mixed references (collected from 1/2 of each of the time course samples) Protocol Name P-MEXP-736 P-MEXP-737 P-MEXP-738 P-MEXP-739 P-MEXP-740 Protocol Type grow specified_biomaterial_action nucleic_acid_extraction labeling hybridization Protocol Description Synchronized fer-15(b26); fem-1(hc17) animals were grown on RNAi bacteria at 25ºC, induced with IPTG on Day 1 of adulthood, and collected from 0-48 hours (10 time points) and 0-196 hours (10 time points); RNAi bacteria was supplemented as necessary for later time points. Worms were floated off lawns with M9 buffer, centrifuged, and washed again in M9. The pelleted worms were dissolved in Trizol (Gibco) and frozen in liquid nitrogen. Hypochlorite-synchronized fer-15(b26); fem-1(hc17) eggs were grown to adulthood on RNAi bacteria at 25ºC, induced with IPTG on Day 1 of adulthood, and collected from 0-48 hours (10 time points) and 0-196 hours (10 time points); RNAi bacteria was supplemented as necessary for later time points. Worms were floated off lawns with M9 buffer, centrifuged, and washed again in M9. The pelleted worms were dissolved in Trizol (Gibco) and frozen in liquid nitrogen. 1. (Before freezing) Add about 2 mL Trizol to 1 mL packed worms, no more than a total of 4 mL, in a 15 mL polypropylene tube (do not use polystyrene, as the Trizol will degrade the tube). Briefly and gently vortex, then quick freeze in liquid nitrogen and store at –80 C. 2. Thaw worm/trizol mixture at room temp; while thawing, add Trizol to 8 mL. Vortex occasionally to help break up the frozen parts and to mix thoroughly. The combination of thawing and vortexing in the Trizol should break open the worms. Let sit an additional 5 minutes to allow more RNA extraction. 3. Spin in Beckman tabletop fuge for 10 min. at 3k rpm. 4. Use a disposable, sterile 10 mL pipette to move the supernatant to a fresh 15 mL tube. Add 1.6 mL of RNAse-free chloroform to each tube; vortex 15 sec, and let incubate 2-3 min. 5. Spin in Beckman tabletop at 3k rpm for 10 min. 6. During the spin, fill 15 mL conical tubes with 6 mL isopropanol. 7. Move the upper (aqueous) layer to the isopropanol tubes; the isopropanol should be in excess, so adjust if the layer was more than 5.5 mL. 8. Incubate in –20 C freezer for 15 min. White precipitate should be visible. 9. Spin in Beckman tabletop for 10 min at 3k rpm. 10. Carefully remove supe; there should be a very visible white pellet. Add 1 mL 75% EtOH in DEPC water; Re-spin for 3 min. 11. Remove supe, let pellets air dry 5-10 min. 12. Dissolve pellets in 1 mL DEPC water. 13. Measure OD 260, 280; freeze at –80 C to store until use. 14. mRNA isolated by Oligotex kit (Qiagen). 15. cDNA reverse transcribed with oligo dT and random primers (pdN6), incorporating amino-allyl dUTP. 16. aadUTP-incorporated cDNA coupled to Cy3 and Cy5 After cDNA synthesis (2 ug polyA RNA with oligo dT and pdN6 primers, aa-dUTP/dNTP), RNA is hydrolyzed (0.5 N NaOH/ 0.25 M EDTA)at 65C, neutralized with Tris-HCl pH 7.4. Tris removed by Microcon or QiaQuick column. cDNA resuspended in 0.05 M Sodium Bicarbonate, pH 9.0. Cy3 or Cy5 monofunctional NHS-ester added to cDNA, let react in dark 1 hr at RT. 4M hydroxylamine added to quench reaction, labeled cDNA purified with QiaQuick PCR purification kit (Qiagen). QiaQuick-purified Cy3 and Cy5 samples paired and mixed, 3 ul 20X SSC, 1.5 ul polyA (10 mg/ml), and 0.5 ul 1M HEPES pH 7.0, then 0.5 ul 10% SDS added. Reaction incubated at 100 C for 2 minutes, then applied to array. Protocol Parameters stop Time;start Time;max temperature;min temperature;media; Amplification;Extracted product; Label used;Amount of nucleic acid labeled;Amplification; Time;Volume;Temperature;Quantity of label target used;Chamber type; Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF SDRF File E-MEXP-9.sdrf.txt Term Source Name The MGED Ontology ncbitax ArrayExpress mo:1.1.3 EFO mo Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version 1.1.3