Retina and RPE/Choroid RNA Extraction Protocol

Chottiwatt Jittprasong

Published: 2023-06-05 DOI: 10.17504/protocols.io.5qpvorjzdv4o/v1

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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.

Before start

Prior to the initiation of this protocol, extraction of the retinal sample from the subject's eyeball is required.

Steps

RNA Extraction

Precool the centrifuge to 4°C

Add 1000µL 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 0h 5m 0s On ice

Add 200µL of Chloroform per 1000µL of TRIzol used for lysis and mix thoroughly.

Incubate for 0h 3m 0s On ice

Centrifuge the sample at 12000x g,4°C

Transfer the aqueous phase containing the RNA to a new tube.

Note

Avoid penetration of the interphase layer.

Add 500µL of Isopropanol to the aqueous phase, per 1000µL of TRIzol used.

Add 4µL of Glycoblue and mix well.

10.

Incubate at -4°C for 1h 0m 0s

11.

Centrifuge at 30000x g,4°C

Citation

Blue pellet should be present at the bottom of the tube.

12.

Discard supernatant carefully.

13.

Resuspend the pellet in 1000µL of On ice 75% ethanol per 1000µL of TRIzol. Vortex and centrifuge at 7500x g,4°C

14.

Repeat Step 13 .

15.

Air dry the RNA pellet for 0h 5m 0s

16.

Resuspend the pellet in 20-50µL of RNase free water.