WormBase Tree Display for RNAi: WBRNAi00102672

WBRNAi00102672 Homol: Homol_homol: F43H9:RNAi
  Sequence_info: PCR_product: sjj_F43H9.1
  Experiment: Laboratory: KQ
    Date: 29 Apr 2008 00:00:00
    Treatment: HT115 bacteria containing each RNAi vector were tested as described previously (Ashrafi et al., 2003) with the following modification: after overnight growth, bacteria were pelleted and resuspended to 5x concentration, 0.1 ml of which was used for seeding each well of a 12-well plate containing 4 ml NGM agar, 6 mM IPTG, and 25 mg/ml carbenicillin. Next, 5-HT or HCl vehicle were added on top of each well, and the plates were allowed to equilibrate overnight. Twenty to thirty synchronized wild-type or mutant L1 animals were placed per well and incubated at 20 degrees Celsius to adulthood. For each RNAi experiment, HT115 (vector alone) and OP50 control wells were also included. All 5-HT suppression phenotypes were confirmed by multiple (5-10) additional rounds of testing on the selected clones, whose identities were confirmed by DNA sequencing. Oxygen consumption was measured in gravid adult animals washed off culture plates with S basal and then washed once more with S basal. For each genotype, 200 animals were deposited in a single well of a 96-well plate containing a biosensor film whose fluorescence intensity increases in proportion to the amount of oxygen consumed (BD Biosciences). Oxygen consumption was measured over a 3 hr period, which we determined empirically to be required for the biosensor film to reach equilibrium. The data reported are endpoint measurements since they accurately reflect changes in oxygen concentration of the samples rather than changes due to equilibration of the biosensor film. Within a single experiment, each genotype was measured in quadruplicate, and each experiment was repeated three to five times. Fluorescence (485 nm excitation, 590 nm emission with cutoff at 550 nm) was measured using a Molecular Devices FlexStation. The mitochondrial uncoupler rotenone (Salway, 1999) (Sigma) decreased oxygen consumption in a dose-dependent manner and was used to validate the assay.
    Delivered_by: Bacterial_feeding
  Inhibits: Predicted_gene: F43H9.1b Inferred_automatically: RNAi_primary
      F43H9.1a Inferred_automatically: RNAi_primary
    Gene: WBGene00001152 Inferred_automatically: RNAi_primary
    Transcript: F43H9.1b.1 Inferred_automatically: RNAi_primary
      F43H9.1a.1 Inferred_automatically: RNAi_primary
  Species: Caenorhabditis elegans
  Reference: WBPaper00031915
  Phenotype_not_observed: WBPhenotype:0002141 Remark: RNAi did not affect the increased oxygen consumption induced by serotonin (5-HT)
      Affected_by: Molecule: WBMol:00004929
  Remark: (Figure 5A) ech-3/F43H9.1 RNAi. Exact sequence used for RNAi not stated by authors, Ahringer laboratory clone used for curation.
  Method: RNAi