6 protocols
Agilent One-Color Microarray Based Gene Expression Analysis Low input Quick amp Labeling. Version 6.5 May 2010
Agilent One-Color Microarray Based Gene Expression Analysis Low input Quick amp Hybridization (v6.5.May 2010)
The array raw data was read and processes by the Feature Extraction software (Agilent) before imported into J-express (http://jexpress.bioinfo.no) for analysis. The data was quantile normalized and missing values replacements were predicted by LS impute Adaptive. All data were log(2)-transformed before downstream analysis.
Data were scanned according to manufacturer specifications.
Salmon lice eggs from L. salmonis inbred through 29 generations were hatched in incubators with flowing seawater and used to infect salmon. Salmon lice were kept in culture on Atlantic salmon (Salmo salar) in tanks with seawater (34.5°, UV treated, 20uM filtered) until adulthood and harvested from hosts. All experiments were conducted in accordance with national animal welfare regulations. Adult female and male lice were collected with forceps from hosts anesthetized with a combination of methomidate (5mg/l) and benzocaine (60mg/l). Samples were recovered by microdissection immediately after sampling or after starvation for 120 hours (starved female intestine only). Organs and tissues were dissected from females in the following sequence; ovaries, intestine, subcuticular tissue and brain enriched tissue. Organs were dissected from males in the following sequence; testis and intestine. Tissues and organs were snap frozen immediately upon dissection and all dissections were performed by the same person. The subcuticular tissue and neuronal and gland enriched tissue (brain) both consist of a variety of cell types whereas the testis, ovaries and intestine are more clearly defined organs.
Total RNA was extracted from the dissected tissues using the RNAeasy Micro kit (Qiagen) according to the Appendix C: RNA Cleanup after Lysis and Homogenization with QIAzol Lysis Reagent. RNA integrity and quantity were measured using the Agilent 2100 Bioanalyzer and NanoDrop Spectrophotometer (OD 260/280 and 260/230 ratios). The RNA samples were frozen at −80°C until analysis.