TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

Total RNA was isolated from retinal tissue by Trizol reagent (Invitrogen), a mono-phasic solution of phenol and guanidine isothiocyanate[37]. The fresh retinal tissue and part of the iris tissue weighing approximately 100 mg was isolated from the donor, frozen in liquid nitrogen, grinded in pre-chilled RNase-free mortars with liquid nitrogen, and then the powder was transferred to a 2 mL RNase-free Eppendorf tube. A volume of 1 mL of Trizol reagent was added into the sample (1 mL Trizol reagent can be used to isolate RNA from 100 mg tissues or 107 cells according to the user's manual from Invitrogen) and mixed. The homogenized sample was incubated at room temperature to permit the complete dissociation nucleoprotein complexes. A volume of 200 μL chloroform per 1 mL Trizol reagent was added and the tube was shaken vigorously by hand for 15 seconds at room temperature for 2 to 3 minutes. The sample was centrifuged at 10 957 × g for 15 minutes at 4°C and the mixture was separated into an upper colorless aqueous phase and a lower red phenol-chloroform phase. The aqueous phase that contained the RNA was transferred to a fresh RNase-free tube. RNA was precipitated in the aqueous phase by mixing 500 μL of isopropyl alcohol at room temperature for 10 minutes. The specimens were centrifuged at 12 000 r/min for 10 minutes at 4°C. After removal of the supernatant, the specimen was kept as a pellet and washed once with 1 mL 75% alcohol and centrifuged at 7 500 r/min for 5 minutes at 4°C. The RNA pellet was dried and dissolved with 50 μL RNase-free water (0.1% diethyl pyrocarbonate treated).

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