Experimental feeding scheme and quantitation of actin expression levels after RNAi treatment in Ephydatia muelleri. A. After feeding for 24 hours with induced HT115(DE3)/L4440-Em-actb or non-induced controls, sponges were washed several times in 1X Strekal's medium, photographed, and harvested for RNA preparation or fixation within 1-2 hour. B. qRT-PCR assay for gene expression levels of Em-actin (purple bar) and two control genes. (Em-six and Em-annexin, green bars) after dsRNA treatment with induced HT115(DE3)/L4440-Em-actb. Levels were normalized to Ef1α and gene expression levels are given as relative levels of RNAi treated sponges against control sponges. Combined data with NEm-actin = 11, NEm-Six = 3, NEm-Annexin = 3; bars represent standard deviations.