Finally, we tested the effect of length of feeding time on the penetrance of RNAi phenotypes observed. Many genes were not effectively silenced after 24 hours at 22°C, but were after 48 hours (see the Materials and methods section and Figure 2). In general, it appears that allowing worms to ingest dsRNA-expressing bacteria for longer periods of time increases the efficiency of RNAi by feeding. However, feeding worms beginning earlier than the L4 stage did not improve the penetrance of RNAi in the progeny (data not shown). After 48 hours, L4-stage hermaphrodites become older adults, and many stop laying fertilized eggs; thus we have routinely used the longest feeding time possible (36-40 hours at 22°C), which ensures that these worms will continue to lay fertilized eggs during the time window from which progeny are assayed (the subsequent 24 hours). We found that 72 hours at 15°C gives a similar level of RNAi inhibition for most genes (data not shown).
