C. elegans RNAi-by-Feeding Knockdown of daf-2 with Kaplan-Meier Lifespan Analysis on FUdR-Treated Plates

N2 worms are age-synchronized by bleaching to obtain L1s. At L4 they are transferred to RNAi plates. Two arms: daf-2 RNAi and L4440 empty-vector control. >=3 independent biological replicates on separate days, n>=80-100 worms/condition/replicate (across multiple plates of ~25-30 worms). Plates contain 100 uM FUdR to prevent progeny. Worms scored every 1-2 days. A sup-35 or known-toxic RNAi can serve as a positive knockdown-efficacy control; an rde-1 mutant (RNAi-deficient) arm controls for RNAi specificity. Scoring is blinded to plate identity.

BSL-1. HT115(DE3) is a non-pathogenic K-12 derivative; handle with standard microbiological technique and decontaminate cultures/plates with 10% bleach or autoclave before disposal. FUdR is a cytotoxic antimetabolite/possible teratogen — wear nitrile gloves, prepare in a fume hood/BSC, and dispose as chemotherapeutic/chemical waste; avoid skin contact and never mouth-pipette. Alkaline hypochlorite (bleach + NaOH) is corrosive — wear goggles and gloves, use in a ventilated area. IPTG and antibiotics handled with gloves. Decontaminate all worm/bacteria waste.

Negative/baseline control: L4440 empty-vector RNAi defines normal lifespan under identical IPTG/carbenicillin/FUdR conditions. RNAi-efficacy positive control: a clone with a visible RNAi phenotype (e.g., dpy-13 or unc-22 twitcher, or pos-1 embryonic lethality) confirms the feeding/induction worked on the day. Specificity control: rde-1(ne219) worms fed daf-2 RNAi should NOT extend lifespan (RNAi-resistant), confirming the effect is RNAi-dependent. FUdR-only and bacteria-only conditions verify FUdR and induction are not independently toxic.

daf-2 RNAi should produce a clear rightward shift in the survival curve, with median lifespan extended roughly 1.5-2x over the L4440 control (e.g., control median ~18-20 days vs daf-2 ~30-40 days at 20 C), and increased maximum lifespan. The rde-1 + daf-2 arm should overlap the control curve (no extension), and the RNAi-efficacy positive control should show its expected visible phenotype, validating knockdown. Censored fraction should stay below ~15%.

• C. elegans N2 (Bristol) wild-type; optional rde-1(ne219) RNAi-deficient control strain
• HT115(DE3) E. coli carrying daf-2 RNAi clone (Ahringer/Vidal library) and empty vector L4440
• NGM-RNAi plates: NGM agar + 1 mM IPTG + 25 ug/mL carbenicillin (+ 12.5 ug/mL tetracycline for HT115 maintenance)
• 100 uM FUdR (5-fluoro-2'-deoxyuridine) in plates
• LB + ampicillin/tetracycline for bacterial culture
• M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4)
• Bleaching solution (alkaline hypochlorite: 0.5 M NaOH + ~1.2% NaOCl) for synchronization
• Platinum-wire worm pick, dissecting microscope

To establish a reproducible C. elegans RNAi-by-feeding lifespan assay knocking down daf-2 from L4 with FUdR-blocked progeny and empty-vector (L4440) control, scoring survival by touch-response and analyzing with Kaplan-Meier/log-rank statistics to quantify median and maximum lifespan extension with proper censoring.

1. Inoculate HT115 daf-2 and L4440 clones in LB+amp+tet, grow overnight at 37 C; back-dilute and grow to OD600 ~0.6.
2. Induce dsRNA: seed 200-300 uL onto NGM-RNAi plates (1 mM IPTG, carbenicillin) and let induce/dry at room temperature overnight, protected from light.
3. Synchronize worms by alkaline-hypochlorite bleaching of gravid adults; let eggs hatch overnight in M9 to arrest at L1.
4. Plate L1s onto OP50 (or directly onto RNAi) and grow to L4 at 20 C.
5. Transfer L4 worms (~25-30 per plate) onto FUdR-containing RNAi plates; this is day 0 of adulthood.
6. Maintain at 20 C. Score survival every 1-2 days by gentle prodding with the pick: no response to touch = dead.
7. Censor worms lost to the wall, bagged (internal hatching), ruptured (vulval explosion), or contaminated.
8. Continue until all worms are dead or censored; record date and fate for each worm.

Independent: RNAi clone identity (daf-2 vs L4440) and strain (N2 vs rde-1). Dependent: individual lifespan (days), median and maximum lifespan, and survival fraction over time. Controlled: temperature (20 C), FUdR concentration (100 uM), IPTG/antibiotic concentrations, food/induction state, worm density per plate, synchronization method, and scoring interval.

Feeding wild-type N2 worms HT115(DE3) bacteria expressing daf-2 dsRNA from the L4 stage will significantly extend median lifespan (expected ~50-100% increase) relative to worms fed empty-vector (L4440) RNAi bacteria, recapitulating the long-lived daf-2 phenotype.

Each worm contributes a survival time and a status (dead vs censored). Survival curves are built by the Kaplan-Meier estimator in OASIS 2 / GraphPad Prism / R survival package. Median lifespan, mean +/- SEM, and percent change vs control are reported per replicate and pooled. Censoring is handled explicitly (lost/bagged/ruptured = censored at last observation). Replicate curves are inspected for consistency before pooling.

1. No lifespan extension with daf-2 RNAi: verify induction (IPTG fresh, plates not too old), confirm with a visible-phenotype RNAi control, and use freshly induced plates. 2) High censoring from bagging/rupture: ensure FUdR is at correct concentration and worms transferred at L4, not earlier. 3) Contamination overgrowing plates: increase carbenicillin, work cleanly, discard contaminated plates and censor affected worms. 4) Variable lifespans between replicates: tightly control temperature (20 C), synchronization, and plate age; score on a fixed schedule. 5) Curves overlap unexpectedly: confirm the daf-2 clone identity by sequencing the insert and check HT115 not lost the plasmid (antibiotic selection).

Survival distributions are compared by the log-rank (Mantel-Cox) test at alpha = 0.05; Cox proportional-hazards regression provides a hazard ratio with 95% CI and allows replicate as a covariate. When multiple conditions are compared, p-values are Bonferroni/Holm-corrected. With n>=80-100/condition across 3 replicates, the design has >90% power to detect a 20% difference in median lifespan. Independent replicate concordance (consistent direction and overlapping HR CIs) is required before reporting.