Abrupt scrib- vs. Abrupt and RasV12 (RasACT) scrib-, NotchICD (NACT) scrib- +/- JNK (Bsk) expression profiles

Drosophila mosaic eye-antennal discs from the listed genotypes generated using the MARCM system were dissected from 3rd instar larvae at day 5 after egg deposition. 20 pairs of discs for the Abrupt and scrib- + Abrupt samples and 50 pairs from FRT control, NACT scrib- +/- BskDN and RasACT scrib- +/- BskDN were used to prepare RNA. Samples were prepared in triplicate, and the RNA isolated using TRIZOL, before being column purified (Qiagen). Probes were hybridized to GeneChip Drosophila 2.0 Genome Arrays (Affymetrix). To compare the expression profile of Abrupt when overexpressed in the eye-antennal discs with tumours formed by Abrupt overexpression in scrib- clones, and to reveal JNK responsive genes in RasV12 (RasACT) scrib- versus NotchICD (NACT) scrib- eye-antennal mosaic discs

采用MARCM（可抑制细胞标记的嵌合分析系统，Mosaic Analysis with a Repressible Cell Marker）系统构建的对应基因型果蝇复眼-触角盘，取自卵产出后第5天的3龄幼虫。其中Abrupt组与scrib- + Abrupt组各取20对眼盘，FRT对照组、NACT scrib- +/- BskDN组及RasACT scrib- +/- BskDN组各取50对眼盘，用于RNA制备。所有样本均设置3次生物学重复，总RNA先以TRIZOL试剂提取，再经Qiagen柱式纯化试剂盒纯化。随后使用Affymetrix公司GeneChip Drosophila 2.0基因组阵列进行探针杂交。本实验旨在对比在复眼-触角盘中过表达Abrupt与在scrib-克隆中过表达Abrupt形成肿瘤时的基因表达谱差异，并揭示RasV12（RasACT）scrib- 相较于NotchICD（NACT）scrib- 复眼-触角嵌合盘中的JNK（c-Jun N端激酶）响应基因。