normalization data transformation protocol
The intensity of probes was extracted from scanned microarray images using Feature Extraction 10.5 software (Agilent Technologies). All algorithms and parameters used in this analysis were the default conditions of the software. Some probes which were judged beyond analysis by Feature Extraction 10.5 software were eliminated from the analysis. ID_REF = VALUE = Lowess-normalized log10 ratio (Cy5/Cy3)
array scanning protocol
Hybridized microarrays were scanned on an Agilent Technologies G2565BA Microarray Scanner System.
A set of fluorescently labeled cRNA targets (750ng for each sample) was employed in a hybridization reaction. Hybridization and washing were performed using the GE Hybridization Kit and GE Wash Pack (Agilent Technologies). The protocols were according to the manufacturer's instructions.
Cyanine-5 (Cy5) or cyanine-3 (Cy3) labeled cRNAs were synthesized from 200 ng total RNA using the Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
nucleic acid extraction protocol
Total RNA was extracted from each of the tail regions by the acid guanidium thiocyanate-phenol-chloroform method.
sample treatment protocol
Approximately 250 Hox10 morpholino(MO) (0.64mM)-injected embryos and control embryos at the tailbud stage (18 hours after fertilization) were cut between the tail and the trunk, and each of the tail regions was collected.
Ciona intestinalis were obtained from the National BioResource Project in Japan.