time post-perforation: 96 hours number of rats per group: 6 sex: male weight: 250-300 grams tissue: tympanic membrane, pars tensa
Treatment protocol
Rats were randomly selected as either controls or in the perforation group. Perforations were created unilaterally (left ear) in the upper outer quadrant of the pars tensa of rats’ tympanic membranes using sterile 23 gauge needles by the first author. Perforations were created while the rat was under general anaesthesia using an inhalational technique with Isoflurane (Oxygen and Isoflurane mixture of approximately 2ml/min Oxygen and 5ml/min (induction) or 2.5ml/min (maintenance) Isoflurane). The tympanic membrane was approached using a 2mm ear speculum using an operating otomicroscope (Zeiss). Before perforation, both ears were examined for signs of inflammation, trauma or disease. No rats were excluded for these reasons. Rats were then randomly allocated into timepoint groups to be sacrificed at either 12, 24, or 36 hours, or 2, 3, 4, 5, 6, or 7 days. At the point of microarray, there were 18 rats per timepoint group and 18 controls. A rat was sacrificed at 12, 24, 36 hours and on day 2, 3, 4, 5, 6, 7. Control rats were also sacrificed. Rats were sacrificed under general anaesthesia (technique above) with 2mls of intracardiac pentobarb. The pars tensa of the tympanic membrane was extracted bilaterally immediately after confirmation of death. To extract the pars tensa, first a postauricular incision was made which was extended to include transection of the ear canal at the bony cartilaginous junction. An incision was then made between the skull base and skin of neck anteriorly allowing exsanguination to occur and provide minimal blood in the field. Soft tissue was dissected off the tympanic bulla using a periosteal elevator and canal skin was removed using a round knife. Using curved needles the pars tensa was dissected from the handle of the malleus and the annulus. Samples were immediately added to tubes containing RNA later and the tubes placed in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
Whenever possible, RNA extraction was completed on the day of tissue collection. If not possible due to time constraints it was extracted as soon as possible. The RNeasy minikit (Qiagen catalog number 74104) was used and the accompanying protocol followed after removing the tubes from liquid nitrogen and removing excess RNA later. All samples were then analysed using a NanoDrop 1000 (Thermoscientific) spectrophotometer to confirm the presence of good quality RNA before sending by overnight courier in dry ice to the Victorian Agribiosciences Centre at the Latrobe University in Bundoora, Victoria. On arrival in Victoria, before proceeding to the pooling stage, samples were again assessed using a NanoDrop 1000 (Thermoscientific) and also an Agilent 2100 Bioanalyzer. The accuracy of the NanoDrop 1000 (Thermoscientific) was first confirmed using 1ug/ul 1kb plus DNA ladder (Invitrogen cat 10787-026). Samples were rejected if they returned an RIN of 6 or less on the Agilent 2100 Bioanalyzer. In cases where the RIN was reported as N/A because of low RNA concentration, the profile was visually inspected and if it contained two obvious peaks similar to other samples in the same timepoint with a passing RIN, these samples also passessed. Samples were randomly pooled into 3 groups (biological replicate groups) per timepoint. There were 18 individuals per timepoint meaning 6 samples were pooled per group. They were pooled so that equal amounts of RNA were taken from each sample. Pooled groups were then assessed using a NanoDrop 1000 (Thermoscientific) and also an Agilent 2100 Bioanalyzer. Pooled groups were rejected using the same criteria as above.
Label
Cy3
Label protocol
The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (version 1.0.1) was followed, which included use of Agilent One Colour RNA Spike-In Kit, cDNA synthesis and amplification, and cRNA purification.
Hybridization protocol
Hybridization was performed according to standard protocols for 17 hours.
Scan protocol
Scanning was performed using an Agilent DNA Microarray Scanner and Agilent's Feature Extraction 9.5.3 Software.
Description
Gene expression in rat tympanic membrane 96 hours after perforation with 23 gauge needle
Data processing
After the microarray data was compiled by the feature extraction software, further analysis was performed using GeneSpring GX9 (Agilent). One microarray (in the day 6 timepoint) was rejected as it appeared to be an outlier in the quality control metrics plot produced by GeneSpring. Further analysis was carried out by GeneSpring on genes identified as either present or marginal. Genes with statistically different expression (p<0.05) between sequential timepoints were identified using a One Way Anova (analysis of variance) test with the Benjamin and Hochberg multiple testing correction and Tukey Post-hoc test.