Strain: Brown Norway rats Age: 6 to 8 weeks TTT-treated Retina, 2-hour
Extracted molecule
polyA RNA
Label
Cy5
Description
A diode laser (IRIS Medical Instruments, Mountain View, CA) emitting at 810 nm wavelength mounted on a modified Haag-Streit slit lamp was used. Laser was applied at 50 mW for 60 seconds, using a 3 mm fixed spot size of this delivery system.Six nonoverlapping laser lesions were delivered to the fundus of each eye covering the visible posterior pole (TTT, transpupillary thermotherapy). Post TTT of 2-hour and also non-treated animals were sacrificed and the eyes were enucleated for RNA extraction. The anterior segment, lens and vitreous were carefully removed and immediately homogenized in TRIzol (Invitrogen, Carlsbad, CA).The total RNA was isolated by the acid guanidine thiocyanate-phenol-chloroform extraction method. Eextracted total RNA was quantified, and 75 μg was used in mRNA purification procedure (Dynabeads mRNA Purification Kit: DYNAL BIOTECH, Oslo, Norway). Dye-labeled cDNA was synthesized from 10 μg of poly(A)+RNA with Direct label Kit (Agilent Technologies, Palo Alto, CA). These dye-labeled cDNA were purified with QIA quick PCR Purification Kit (QIAGEN) and then hybridized with rat cDNA microarray. The microarray plates were scanned by a Microarray Scanner (Agilent Technologies) to detect and quantify the dye-signal intensity of each well.
Data processing
Local background correction and LOWESS normalization was accomplished by Feature Extraction Software (Agilent Technologies).
