Jun 05, 2023
Retina and RPE/Choroid RNA Extraction Protocol
- 1City University of Hong Kong
Protocol Citation: Chottiwatt Jittprasong 2023. Retina and RPE/Choroid RNA Extraction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorjzdv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
In Use
Created: June 04, 2023
Last Modified: June 05, 2023
Protocol Integer ID: 82864
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Abstract
This protocol involves the extraction of RNA from retinal and retinal pigment epithelium (RPE) cells in the subject's retina, utilizing TRIzol as the main reagent for cell lysis.
Safety warnings
TRIzol is extremely corrosive and toxic. Exercise extreme caution while handling TRIzol (e.g. handling within the fume hood).
Before start
Prior to the initiation of this protocol, extraction of the retinal sample from the subject's eyeball is required.
RNA Extraction
RNA Extraction
1h 48m
1h 48m
Precool the centrifuge to 4 °C
Add 1000 µL of TRIzol per retina and pipette up and down 30 times to homogenize the mixture.
Safety information
This step should be done in fume hood due to toxicity of TRIzol.
Incubate for 00:05:00 On ice
5m
Add 200 µL of Chloroform per 1000 µL of TRIzol used for lysis and mix thoroughly.
Incubate for 00:03:00 On ice
3m
Centrifuge the sample at 12000 x g, 4°C, 00:15:00
15m
Transfer the aqueous phase containing the RNA to a new tube.
Note
Avoid penetration of the interphase layer.
Add 500 µL of Isopropanol to the aqueous phase, per 1000 µL of TRIzol used.
Add 4 µL of Glycoblue and mix well.
Incubate at -4 °C for 01:00:00
1h
Centrifuge at 30000 x g, 4°C, 00:15:00
Expected result
Blue pellet should be present at the bottom of the tube.
15m
Discard supernatant carefully.
Resuspend the pellet in 1000 µL of On ice 75% ethanol per 1000 µL of TRIzol. Vortex and centrifuge at 7500 x g, 4°C, 00:05:00
5m
Repeat Step 13.
Air dry the RNA pellet for 00:05:00
5m
Resuspend the pellet in 20-50 µL of RNase free water.
Note
40 µL of RNase free water is recommended.
Quantify the sample by NanoDrop