Retina and RPE/Choroid RNA Extraction Protocol

Chottiwatt Jittprasong

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

Disclaimer

DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK

The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.

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.

Note

40µL of RNase free water is recommended.

17.

Quantify the sample by NanoDrop

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing