Tissue preparation protocol. Animal were killed by rapid cervical dislocation. Retinas were removed immediately and placed in RNAlater at room temperature. Two retinas from one mouse were stored in a single tube.

Each array was hybridized with a pool of cRNA from 2 retinas (1 mouse). Natalie Freeman-Anderson extracted RNA at UTHSC.

 

Dissecting and preparing eyes for RNA extraction

 

Retinas for RNA extraction were placed in RNA STAT-60 (Tel-Test Inc.) and processed per manufacturer’s instructions (in brief form below). Total RNA was extracted with RNA STAT-60 (Tel-Test Inc.) according to the manufacturer's instructions. Briefly we:

 

• Homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue via syringe)
• Allow the homogenate to stand for 5-10 min at room temperature
• Add 0.2 ml of chloroform per 1 ml RNA STAT-60
• Mix the sample vigorously for 15 sec and let the sample incubate at room temperature for 5-10 min
• Centrifuge at 12,000 g for 1 hr at 4°C
• Transfer the aqueous phase to a clean centrifuge tube
• Add 0.5 ml of isopropanol per 1 ml RNA STAT-60
• Vortex and incubate the sample at -20°C for 1 hr or overnight
• Centrifuge at 12,000 g for 1 hr
• Remove the supernatant and wash the RNA pellet with 75% ethanol
• Remove ethanol, let air dry (5-10 min)
• Dissolve the pellet in 50 μl of nuclease free water.