Table 2. Evaluation of the assay system 2: lac2 as the marker gene.

1st RNAi	2nd RNAi		Initial # of larvae	# survived	# of 2nd instar	lac2	WT	suppression
250 bpAgo2 (5 ug/mL)	250 bp lac2 (5 ug/mL)	co-feeding	30	26	19	18	1	5%
250 bp Dcr2 (5 ug/mL)	250 bp lac2 (5 ug/mL)	co-feeding	28	21	21	17	4	19%
250 bp KA (5 ug/mL)	250 bp lac2 (5 ug/mL)	co-feeding	27	24	24	17	7	29%
250 bpAgo2 (5 ug/mL)	250 bp lac2 (5 ug/mL)	sequential	30	22	16	15	1	6%
250 bpDcr2 (5 ug/mL)	250 bp lac2 (5 ug/mL)	sequential	28	13	12	12	0	0%
250 bp KA (5 ug/mL)	250 bp lac2 (5 ug/mL)	sequential	27	20	15	11	4	26%

The amount of the maker gene dsRNA and the feeding scheme influence the outcome of the assay. The second instar larvae were categorized into the Lac2 phenotype (i.e. the second RNAi worked) and the WT phenotype (i.e. the second RNAi is suppressed). The suppression of the second RNAi phenotype by the initial RNAi was evaluated as the proportion of the WT larvae in the survived second instar larvae (Suppression).