RNA can be extracted from a variety of tissues and used to sequence cDNAs (using 3' or 5' RACE) or to quantify relative gene expression (real time RT PCR). While some protocols isolate mRNA (using a polyT purification column), we haven't bothered with this additional step as the mRNA we are interested in can be selected at the reverse transcription step.

Recently we have switched to Qiagen RNeasy kits. These are quite a bit expensive (about $5/each) but the RNA is reliably high quality. This protocol is given here:

RNeasy.

The protocol for using Trizol is described below. While it is very inexpensive, it involves an isopropanol precipitation. This has two problems: 1) the pellet can be lost, 2) the pellet can be hard to redissolve.

Trizol

For retinal studies, euthenize the fish using MS-222. Remove the eyes. For larval fishes, the entire eyeball can be used. For adults (eyes > 4 mm diameter), hemisection the eye and tease out the retina (cutting the optic nerve at the back to release it).

At this point, the tissue can be place in RNAlater (Ambion) and put at 4C overnight. Then the next day it should be transferred to -20C. At a later date, the RNAlater can be pipetted off an replaced with Trizol to continue the following procedure.

Alternatively the tissue can be immediately placed in 0.5 ml of trizol in a 1.5 ml micropestle tube (VWR KT749520-0090) and homogenized. Following this, the tissue can be sheared by several passes through a 21-22 gauge syringe needle. Then 0.5 ml more of Trizol is added and mixed with the syringe. For very small eyes, 10 ug of glycogen 10 ul of 1 mg/ml RNAse free stock) can be added at this point to increase the yield.

If processing several samples, samples can be left at this point (for up to several hours) until all samples are done.

Add 0.2 ml chloroform (for each ml of Trizol used). Shake for 15s to mix well and incubate for 2-3 minutes. Centrifuge for 15 min at 12000g and 4C. At this point a clear aqueous layer should be on top and a red layer on the bottom. However, sometimes, there is a fair bit of pink to the top layer. This usually still gives good RNA.

Transfer the aqueous layer to a clean, RNAse free tube. Take care to pipette slowly as there is considerable material at the aqueous / organic interface that is easy to take up. Add 0.5 ml isopropanol. You can now leave this at 4C overnight (but not at -20C). Alternatively, incubate at room temp for 10 min, then spin 10 min at 12000g and 4C. You should now see an RNA pellet.

Remove iPrOH. Wash pellet with 1 ml (per ml of Trizol) of 75% ethanol. Spin 5 min at 7500g and 4C. Remove EtOH being careful not to remove pellet (which sometimes comes loose at this point). Air dry for 10 minutes (don't speed vac). Add 50 ul RNase free water. Store at -80C.