80
solution was vortexed, and allowed to incubate at room temperature for 2 minutes. Then the solution was centrifuged at top speed in an eppendorff microcentrafuge for three minutes, and the aqueous layer containing the retinal RNA was removed. The RNA was precipitated by adding an equal volume of isopropanol (Fischer Scientific; Fair Lawn, NJ) to the aqueous phase, incubating five minutes at room temperature, and spinning at maximum speed in an eppendorff microcentrafuge. The RNA was then washed two times with cold 70% ethanol, dried, and resuspended in 20 p.L of sterile water.
Approximately 6 ig of total RNA extract was incubated with 600 nM ribozyme and placental RNase inhibitor (Promega, Madison, WI) to prevent degradation by residual cellular RNases in cleavage buffer at 37 oC. Aliquots were removed from the reaction at 0, 6,12,24,and 48 hours. Absolute ethanol was added to the aliquots to stop the cleavage reaction and precipitate the RNA.
RT-PCR Reaction
The aliquots were ethanol precipitated, washed twice with cold 70% ethanol, and dried. A reverse transcription reaction was started by resuspending the cleavage reaction in 11 jtL of sterile water and a 1 [tL solution containing 2 pM of an antisense P-actin primer and 1 pM of an antisense mouse opsin primer. This mixture was heated at 70 oC for 10 minutes and quick chilled on ice. Next, 4 gL of 5x buffer (250 mM Tris-HCI pH
8.3, 2 gL 0.1 M DTT, 1 gL 10mM dNTP) was added to each reaction. Following a 5 minute incubation at 42 oC, 1 tL of Superscript II (Life Technologies; Grandview, NY) was added and the reaction was incubated for 50 minutes at 42 oC. The product of the reverse transcription reaction was amplified by a 25 cycle polymerase chain reaction (PCR) reaction in the presence of 60 pM of 3-actin primer and 30 pM of opsin specific