Optimization of planarian fixation for RNA extraction and histology

Trizol extraction of total RNA from planarians

(based on Invitrogen's protocol)

Notes: When working with RNA, maintain an RNAse-free work area, and use RNAse-free tubes, pipets, and solutions etc. at all times. For tips on good practices, see:

a. Green, M.R., and Sambrook, J. (2012). “Winning the battle with RNAses.” In Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp. 450-51.

b. https://www.thermofisher.com/us/en/home/references/ambion-tech-support/working-with-rna-the-basics.html

Extreme care should be taken with phenol, which is corrosive. A small droplet can cause severe skin damage, therefore gloves, goggles, and a lab coat are required. Inhalation of vapors should be avoided. Guanidine isothiocyanate is also moderately toxic. All Trizol/phenol/chloroform waste should be properly disposed of. For further info refer to the Cold Spring Harbor Molecular Cloning manual and the Trizol MSDS and protocol sheets.

Homogenization

1. Place 5 large asexual animals (8-10 mm, ~9 mg ea. – Invitrogen recommends starting with 50-100 mg tissue) into an RNAse-free 1.5ml eppendorf tube, then remove all salts.

2. Add 200 µl Trizol reagent.

3. Homogenize using small blue RNAse-free Kontes micro-pestle (20-30 strokes, or 20-30 sec with the automated homogenizer). Use extreme care not to splash phenol on your skin or anywhere else. If possible, perform this step in a chemical hood, aiming the tube opening away from yourself, and using the sash to protect your upper body.

4. Add 800 µl Trizol, pipet gently or invert to mix.

5. Allow to incubate for 5 min at room temperature (RT), then gently pipet again. Most tissue will solubilize but some gooey clumps will remain.

6. Proceed with optional centrifugation (12,000 x g, 10 min, 4ºC) to remove insoluble material.

7. Transfer supernatant to a fresh tube.

Phase Separation

8. Add 0.2 ml chloroform, make sure eppendorf cap is completely closed, shake vigorously for 15 sec, then incubate for 3 min at RT. Next centrifuge at 12,000 x g, 15 min, 4ºC. After phase separation the aqueous (top) layer will be slightly grayish, and lots of proteinaceous debris will be at the interphase.

9. Transfer upper/aqueous phase to a fresh tube, ~450-600 µl. NOTE: it is much better to leave ~100 µl of aqueous phase behind than to try to recover every last drop. RNAses are usually abundant in the interphase, and phenol contamination of the aqueous phase/RNA will interfere with downstream applications.

10. Second chloroform extraction: add 500 µl chloroform, shake vigorously for 15 sec, then incubate for 3 min at RT. Next centrifuge at 12,000 x g, 15 min, 4ºC.This step removes residual phenol, and is especially important when low RNA yields (<10 µg) are expected, for example from fewer or smaller animals.

11. Transfer upper/aqueous phase from second extraction to a fresh tube.

• Optional: If low RNA yields (<10 µg) are expected, add RNAse-free glycogen (10 µg, see Invitrogen Trizol protocol) as a carrier to increase yield.

Precipitate

At this point the optional precipitation (Invitrogen protocol) for samples rich in proteoglycans/polysaccharides is utilized. This step precipitates RNA while leaving pigments/mucus/etc. in solution.

12. Add 0.25 ml isopropanol plus 0.25 ml high salt precipitation solution (0.8M sodium citrate + 1.2M NaCl (DEPC-treated)), mix by inversion, incubate at RT for 10 minutes, then centrifuge 12,000 x g, 10 min, 4ºC. From 5 planarians, a clearly visible, whitish pellet should be visible after the spin.

13. Remove as much supernatant as possible (i.e., remove most, quick spin, and remove the rest with a P100 tip). The salt (from the high salt solution) will precipitate in the next step otherwise.

Wash & Dry

14. Add 1 ml ice-cold 75% ethanol (made with DEPC-treated water). OPTIONAL: vortex 5-10 seconds to break up the pellet, then centrifuge 7500 x g, 5 min, 4ºC.

15. Remove as much ethanol wash as possible without disturbing the pellet, then air dry just until the pellet begins to clear. This usually takes 1-5 min. Do not overdry, as this makes RNA difficult to resuspend.

Redissolve

16. Add 50 µl RNAse-free water, flick to mix. Do not pipet, since RNA pellet can stick to the pipet tip and be lost. Pellet will dissolve at RT in a few minutes, but sample can be heated to 50ºC for 5 min if desired.

17. Spectrophotometric analysis: quality RNA has an A₂₆₀/A₂₈₀ ratio of 2.0-2.2. 2.3 and above indicates degradation, less than 1.6 indicates contamination and/or partial resuspension. From 5 large animals, 50-100 µg RNA (i.e., 1-2 µg/µl) is a typical yield. 

18. Proceed immediately with DNAse treatment or store RNA at -80 ºC.

• Optional: If pellet is a smear on the side of the tube, the tube can be briefly vortexed, then spun down, to be sure all RNA is dissolved.

Products

TRIzol Reagent: ThermoFisher 15596026

Kontes Pestles:  FisherK749521-0590

Automated Cordless Pestle Homogenizer: Fisher FS7495400000