Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) . We assessed the gene expression levels of two transcription factors, NF- κ B1 (nuclear factor kappa B1) and AP1 (activating protein 1). AP1 comprises c-FOS and c-JUN, and these genes were assessed relative to glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) using qRT-PCR; primer sequences are shown in Supplemental Material, Table S2 (http:////). Total RNA was isolated from the whole head regions of treated and untreated embryos ( n = 3 embryos/group) using an RNeasy® Mini Kit (QIAGEN, Valencia, CA). RNA was quantified with the NanoDrop TM 1000 Spectrophotometer (Thermo Scientific, Waltham, MA), and integrity was verified with a model 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). Fold changes between the treatment group and the control group were calculated using delta-delta cycle threshold (ΔΔCt) values and normalized with GAPDH as a housekeeping gene. Statistical significance of the transcript levels between treatment groups versus the control group was calculated using an unpaired t- test.