tissue: Inner ear treatment: 2 days after sound exposure
Treatment protocol
RNA samples were obtained from the inner ears of the three groups of 18 to 20 fish each (controls, 2 dpse, 4 dpse). One group served as non-sound exposed controls, and the remaining two groups were exposed to the acoustic stimulus and allowed to recover for 2 or 4 days. The day 2 time point was selected in order to investigate gene expression during proliferation, which had been shown to peak at 2 dpse, and 4 dpse was chosen since it represented a post-proliferative phase
Extracted molecule
total RNA
Extraction protocol
Fish were sacrificed one at a time with an overdose of MS-222, their heads were removed, and both whole ears (saccule, lagena, utricle and semi-circular canals) were immediately dissected out while being completely submerged in RNAlater (Ambion, Austin, TX), as preliminary work indicated that either the small size of the saccule, or the length of time needed to separate it from the inner ear, resulted in low RNA yield. Ears were then placed in sterile Eppendorf tubes and flash frozen in liquid nitrogen. Three to four hours were required to dissect all the fish in one group. Once all the ears for a sample were collected, the tissue was pooled and homogenized with a Kontes Pellet Pestle Microgrinder and sterile disposable pestles (Kontes, Vineland, NJ), then processed for RNA isolation using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA). RNA quality was checked with the aid of an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE). For this project, sharp ribosomal RNA bands were evident with an RNA integrity number greater than 7.0.
Label
Cy3
Label protocol
300 ng total RNA was used to generate fluorescent cRNA with the aid of Low RNA Input Linear Amplification kit with one-color (Agilent, Wilmington, DE). Briefly, this kit uses a T7 promoter primer to synthesize cDNA and T7 RNA polymerase to synthesize cRNA, which simultaneously amplifies the target material and incorporates cyanine 3-labeled CTP (Cy3). The labeled cRNA was purified by using the RNeasy Mini Elute kit (Qiagen, Valencia, CA). The yield and incorporation efficiency were measured on a spectrophotometer (NanoDrop Technologies). The yield for this project was greater than 1.5 ug, and the specific activity was greater than 9.0 pmol Cy3 per ug cRNA.
Hybridization protocol
1.65 µg of each labeled cRNA sample was fragmented at 60 °C for 30 min (Agilent Gene Expression Hybridication kit) and then hybridized to Agilent Zebrafish (Danio rerio) oligonucleotide arrays (Agilent Unrestricted AMADID Release GE 4x44K, 60-mer oligonucleotides; G2519F; V1: 015064) at 65 °C for 17 hours. This microarray has 21,000 D. rerio probes, replicated twice. Three technical replicates were hybridized for each of the three time points (control, Day 2, and Day 4), with one replicate of each time point on each of the three 4-array plates processed. After hybridization, the microarray slides were washed with Agilent gene expression wash buffers.
Scan protocol
The slides were scanned with the aid of an Agilent microarray scanner (G2565BA) with a setting for one-color using the green channel and 5 µm resolution.
Description
Gene expression 2 days after sound exposure
Data processing
The one-color microarray images (.tif) were extracted with the aid of Feature Extraction software (v 9.5.1, Agilent). The raw data were normalized and then filtered by flag using GeneSpring software. Normalization: 1. log2 transformation 2. Per chip: normalized to 50% quantile 3. Per gene: Normalized to median. Filtered on the Flag: Entities (genes) were filtered based on their flag values. Flag values were selected that an entity must satisfy to pass the filter by defining the acceptable flags (Present or Marginally Present). We retained entities in which at least 3 out of 9 samples had acceptable values.
