tissue: skin gender: male location: Mississippi Sound collection date: August 15 2011 season: Summer
Extracted molecule
total RNA
Extraction protocol
Samples were obtained by remote dart biopsy using the method of Hansen et al. (2004), employing a modified 0.22-caliber rifle affixed with a video camera to collect images of dorsal fins for individual identifications. The rifle fired a bolt which consisted of a thin-walled, hollow arrow shaft and a hollow cylindrical collecting head approximately 2.5 cm in length and 1.0 cm in diameter. Each sample was a cylindrical shaped core of skin and blubber, weighing approximately 0.5–1.0 g. The sampling location on the body of the dolphin varied to some degree between individuals, but all the samples were collected within an area that measured approximately 50 cm x 30cm centered below the dorsal fin above the lateral midline. Prior to each use, the collecting head was scrubbed, soaked in a 10% bleach solution, then rinsed twice, first with distilled water and then with ethanol. Retrieval of the sample in the collecting head, affixed to a float, was carried out by reel. Once on deck, the sample was removed from the sampling tip with sterile forceps and scalpel, then transferred to a cryovial and snap frozen and stored in liquid nitrogen until transfer to the laboratory. In the laboratory, samples were they were stored under chain of custody at -80°C until processing Approximately 0.05-0.15 g of skin was dissected from frozen integument biopsies using a scalpel, on dry ice, just medial to a white line demarking the fibrous layer of the dermis and further cut into pieces ≤ 0.2 cm thick. The dissected sample was transferred to 1.5ml of RNAlater Ice (Ambion) that was prechilled to -80°C and soaked for at least 16 hours to transition tissue from -80 °C to - 20°C prior to processing. The sample was then removed from the RNAlater ice and placed in a 2.0 ml microcentrifuge tube containing 1ml Qiazol (Qiagen) and a 5mm stainless steel bead, and homogenized 3x using a Qiagen Tissuelyser at 20 Hz for 3 min. The sample was cooled on ice for 30 sec between sets. The homogenate was transferred to a new 2.0ml microcentrifuge tube and incubated on the benchtop at room temperature for 5 minutes before adding 200µl of chloroform. The tube was shaken vigorously for 15 seconds then incubated on the benchtop at room temperature for 3 minutes. After 15 min centrifugation at 12,000 x g 4°C, 500 µl of the upper aqueous phase was transferred to a new 1.5ml microcentrifuge tube containing 500 µl of 70% ethanol, mixed by pipetting, and then immediately transferred to a Qiagen RNeasy spin column. The protocol for Purification of Total RNA using the RNeasy Lipid Tissue Mini Kit with on-column DNase digestion (Qiagen RNeasy Lipid Tissue Handbook 02/2009) was followed exactly from this point on with a 60µl final elution in RNase-free water. The RNA quantity determined using a Nanodrop ND-1000 spectrophotometer and quality was assessed using an Agilent 2100 Bioanalyzer. Only samples with an RNA integrity number (RIN) of > 7.0 were used in the study.
Label
Cy3
Label protocol
Total RNA (50ng) was amplified and Cy 3 labeled using Agilent’s Low Input Quick Amp Labeling Kit. The Cy3 labeled cRNA was quantified using a Nanodrop ND-1000.
Hybridization protocol
A total of 1.65 mg of Cy3 labeled cRNA was hybridized to the microarray. The One-Color Microarray-Based Gene Expression Analysis Protocol (Version 6.5, May 2010) was followed for both the labeling and the hybridization. Hybridization was carried out at 65°C in an Agilent hybridization oven rotating at 10rpm for 17 hours.
Scan protocol
Slides were scanned with an Agilent G2505B scanner equipped with Agilent Scan Control software, using the Extended Dynamic Range Scan Mode, 5µM scan, XDR PMT Hi 100% Lo 10%. Images were extracted using Agilent Feature Extraction version 10.7.3.1.
Data processing
Text (.txt) files obtained from Agilent Feature Extraction (FE) were imported into Agilent’s GeneSpring 11.5.1 software and processed as Agilent single color arrays. Normalized signal values were generated after thresholding the raw signals to 1.0, summarization (summarization is performed by computing the geometric mean), and log transformation, followed by 75th percentile shift normalization.
