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<blockquote>
<p><strong>Tissue preparation protocol</strong>. Animal were killed by rapid cervical dislocation. Retinas were removed immediately and placed in RNA<em>later</em> at room temperature. Two retinas from one mouse were stored in a single tube.</p>
<p>Each array was hybridized with a pool of cRNA from 2 retinas (1 mouse). Natalie Freeman-Anderson extracted RNA at UTHSC.</p>
<p> </p>
<p><strong>Dissecting and preparing eyes for RNA extraction</strong></p>
<p> </p>
<p>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:</p>
<p> </p>
<ul>
<li>Homogenize tissue samples in the RNA STAT-60 (1 ml/50 to 100 mg tissue via syringe)</li>
<li>Allow the homogenate to stand for 5-10 min at room temperature</li>
<li>Add 0.2 ml of chloroform per 1 ml RNA STAT-60</li>
<li>Mix the sample vigorously for 15 sec and let the sample incubate at room temperature for 5-10 min</li>
<li>Centrifuge at 12,000 g for 1 hr at 4°C</li>
<li>Transfer the aqueous phase to a clean centrifuge tube</li>
<li>Add 0.5 ml of isopropanol per 1 ml RNA STAT-60</li>
<li>Vortex and incubate the sample at -20°C for 1 hr or overnight</li>
<li>Centrifuge at 12,000 g for 1 hr</li>
<li>Remove the supernatant and wash the RNA pellet with 75% ethanol</li>
<li>Remove ethanol, let air dry (5-10 min)</li>
<li>Dissolve the pellet in 50 μl of nuclease free water.
<p> </p>
</li>
</ul>
</blockquote>
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