RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 104

Scoring Enhancement by Visual Inspection
We first examine the images of the ts mutant fed on the L4440 empty vector
control. We typically inspect at least 48 images to gain a sense of the variation in
number of adults and progeny (larvae/eggs) per well in the absence of any RNAi
effect. Usually, a ts mutant strain fed on the L4440 control at semi-permissive
temperature will produce numerous larvae and few eggs.
Next, we score the 96 images of the first double-knock-down plate (plate A),
comparing always with what is expected from the L4440 control. Any wells that
resemble the ts mutant fed on the L4440 control are labeled ''not enhanced'' (NE).
We note any differences in viability of the progeny and brood size, recording
Embryonic lethality (Emb), Sterility (Ste), and deviations in the number of larvae
and eggs. We also score any detectable post-embryonic phenotypes (including larval
arrest (Lva), larval lethality (Lvl), Growth arrest (Gro), ruptured worms (Rup),
protruding vulva in adults (Pvl), and egg laying defective (Egl). We then score the
96 images of the second test plate for the same double-knock-downs (plate B) in the
same way.
From the scores of plates A and B, any image with a phenotype deviating
from the L4440 control is compared to the corresponding image of N2, which
was fed on the same RNAi bacteria. If the N2 RNAi looks WT, then we score
the target gene in the double-knock-down as a putative enhancer, which we
place into one of the four categories: we score a result as ''strong'' if it shows
Emb, Ste, or a highly reduced number of larvae (VLL); as ''medium'' if it shows
a higher proportion of unhatched embryos ( > E, typically signifying incomplete
penetrance lethality) or a smaller brood size (identified as fewer larvae (LL) or
less embryos ( < E) depending on the screening temperature, signifying either
incomplete sterility or reduced fertilization); as ''weak'' if the N2 control shows
some RNAi effect or if no conclusive score can be assigned (?); or as ''post-
embryonic enhancement'' (PEE) if we detect any post-embryonic phenotypes
not present in the N2 RNAi. Fig. 5 shows an example of an enhancement
interaction.
Once we obtain a score for both replicates from each experiment (copies A and B),
we perform secondary screening of all putative strong, medium, and post-embryonic
enhancers identified in at least one replicate and of putative weak enhancers iden-
tified in both replicates.
Scoring Suppression by Visual Inspection
As for scoring enhancement, we first examine 48 images of the ts mutant fed on
control vector L4440 to gain a sense of the variation among the wells. Usually, a ts
mutant strain fed on the L4440 control at semi-restrictive temperature will produce
numerous eggs and few larvae, if any.
Next, we score the images from both experimental replicates in comparison with
the expected phenotype from the control L4440 RNAi plate. We record results with