strain: AB (wild type) gender: Female age: 4-12 months
Biomaterial provider
Texas A&M Zebrafish Facility
Treatment protocol
Reproductive males and females (3 each) were paired on day 1, and spawning was confirmed on day 2 by the presence of eggs. Males and females were subsequently separated and housed for five days. On day 7 individuals were sacrificed and dissections were performed.
Growth protocol
Standard rearing protocols were used, as described in Westerfield, 2000: Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.
Extracted molecule
total RNA
Extraction protocol
Standard Trizol (Invitrogen) extraction.
Label
biotin
Label protocol
Standard Affymetrix
Hybridization protocol
Standard Affymetrix
Scan protocol
Standard Affymetrix
Description
Upon euthanasia the entire reproductive tract from each individual was removed under a dissecting microscope (320X) with sterilized fine-tipped forceps, placed in 200 ul Trizol, and snap-frozen in a dry ice-ethanol bath. At this time the remainder of the body was placed in 4000 ul Trizol, and similarly snap-frozen. All downstream procedures were performed according to standardized manufacturer protocols.
Data processing
Data were analyzed 4 different ways:
VALUE [GCOS]: Expression values were computed using the MAS5 algorithm in GCOS version 1.2, under suggested settings. All probesets were scaled using a TGT value of 500, and no normalization was applied (i.e., the normalization factor was 1.0).
VALUE2 [GC-RMA]: Expression values were computed using the GC-RMA model-based algorithm in GeneSpring GX 7.3.1 (Agilent Technologies). Default normalization is performed as follows: the raw intensity value for each probe set on a chip is divided by the overall median intensity value for that chip. This “prenormalized” value is then divided by the median “prenormalized” value for that specific probe set across all 12 chips in the experiment.
VALUE3 [PM-Only]: All 12 chips were analyzed simultaneously in dCHIP, which uses a model-based algorithm to estimate expression values. The normalization procedure implemented in dCHIP normalizes all arrays in the analysis against the array with median intensity. This array was analyzed using the PM-Only procedure under default parameters.
VALUE4 [PM-MM]: All 12 chips were analyzed simultaneously in dCHIP, which uses a model-based algorithm to estimate expression values. The normalization procedure implemented in dCHIP normalizes all arrays in the analysis against the array with median intensity. This array was analyzed using the PM-MM procedure under default parameters.
