ID_REF = VALUE = normalized signal intensity
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) to obtain background subtracted Processed Signal intensities.
Slides were scanned immediately after washing on the Agilent DNA Microarray Scannerusing one color scan setting for 8x15k array slides.
600 ng of purified Cy3-labeled cRNA was hybridized to the custom 8x15,000 G. holbrooki microarray using Gene Expression hybridization kit (Agilent, Santa Clara, USA). In brief, Cy3 was incubated with blocking agent and fragmentation buffer at 60 oC for 30 minutes. Hybridization buffer was then added to the sample and 40 μL of the sample was added to the gasket. The microarray slide was then incubated with the gasket for 17 hours at 65 C while spinning at 10 rpm. The slide was then washed and dried.
For amplification and labelling, the Quick Amp Labeling kit (Agilent, Santa Clara, USA) was utilized with adjustments made for a half reaction, one color (Cy3) protocol. In brief, T7 primers were annealed to 1000 ng RNA with the addition of RNA spike-in controls (One color RNA spike-in mix; Agilent, Santa Clara, USA) and were incubated at 65 oC for 10 minutes. cDNA synthesis using MMLV-RT was conducted at 40 oC for 2 hours followed by 65 oC for 15 minutes. cDNA was reverse-transcribed into cRNA using a T7 RNA polymerase with the addition of Cy3 to the reaction for incorporation into the samples. in vitro transcription proceded at 40 oC for 2 hours. Labeled cRNA was purified using the RNeasy kit (QIAGEN, Hilden, Germany) and eluted in nuclease-free water. The cRNA concentration and specific activity (pmol Cy3 per µg cRNA) was determined using the Nanodrop (ThermoScientific, Waltham, USA).
G. holbrooki from laboratory stocks were transferred to aerated 7L glass tanks and exposed via semi-static renewal (60% water change daily) to either the vehicle control (ethanol, 0.0014% final concentration) or 1 μg/L of the potent androgen receptor (AR) agonist TB (17β-hydroxyestra-4,9,11-trien-3-one) (Steraloids, Newport, USA). Four tanks per treatment were utilized and two G. holbrooki were sampled from each tank in both treatment groups after 14 days of exposure. Mosquitofish were anesthetized using Tricaine-S (Western Chemical, Ferndale, USA) and sacrificed via spinal transection. Livers were removed, snap-frozen in liquid nitrogen, and stored at -80 oC until RNA isolation.
RNA was isolated from frozen liver samples using TRIzol reagent (Invitrogen, Grand Island, USA) following the manufacturer’s protocol. In brief, livers were homogenized in 1 mL TRIzol and after a 5 min incubation at room temperature, 200 µL chloroform was added and the upper aqueous layer containing the RNA was separated from the organic layer via high-speed centrifugation. The RNA was precipitated out of the aqueous layer using isopropanol and washed twice with ethanol. The quality and quantity of the RNA was determined using the Nanodrop (ThermoScientific, Waltham, USA) as well as the 2100 BioAnalyzer (Agilent, Santa Clara, USA).