Worm Breeder's Gazette 12(3): 15 (June 15, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
As Dr. R. Horvitz's laboratory asked us for a good protocol for RNA extraction from Ascaris eggs and embryos, other labs might also be interested in this method. The problem with Ascaris is that it has a very rough egg shell; that's why it is rather difficult to obtain RNA from different developmental stages. With the method described below, one can isolate it efficiently. This RNA can be used to perform Northern blots, polyA+ isolation, c-DNA cloning, etc.
Collection of eggs from Ascaris lumbricoides var. suum
-Decomposition of the uteri and removal of the proteinatious layer of the eggs are done by incubating the uteri overnight at 4°C in 0.5 N NaOH with gentle agitation with a magnetic stirrer.
-Let the eggs settle down (ca. 1 hour) or centrifuge them in a swing-out rotor at 7009 for 2 min. Rinse the eggs 5 times in 10 volumes of tap water. At this stage, the eggs are resuspended in 0.1 N H2S04 and can be stored at 4°C for days, months or even years!
To obtain different embryonic stages, one incubates the eggs for the desired time with 10 volumes of 0.1 N H2 SO4at 30°C in a large erlenmeyer; the latter, closed by a paper towel, is gently agitated in a rotatory shaker.
Removal of the chitinous layer
-Resuspend a pellet of eggs in 5 vol. 3.8% NaClO during 1 min. in a 50 ml Falcon tube.
-Centrifuge in a swing-out rotor at 7009 during 1 min.
-Resuspend the eggs in 5 vol. 3.8% NaClO by inverting the tube 3-4 times.
-Leave the tube in a vertical position for 10 min. The eggs should now swim on top of the solution.
-Add 1 vol of water and mix.
-Centrifuge in a swing-out rotor at 7009 during 1 min.
-Rinse the eggs 5 times in 10 volumes of distilled water.
Note: between the second and third rinse the non-fertilized eggs burst, giving a white color to the water.
RNA extraction:
This method is based on Chomczynski's protocol (Analytical Biochemistry 162 (1987), 156-159)
-Resuspend the eggs (ca.0.7 ml of packed eggs) in 3 ml of solution D: 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol.
-Homogenize the eggs in a teflon-glass homogenizer with 1 mg of glass beads (diameter 0.1 mm); 3-4 strokes are sufficient.
-Check whether the lysis of the eggs is complete with a microscope
-Transfer the solution into a 15 ml Falcon tube.
-Add 0.3 ml of 2 M NaAc (pH 4) and mix.
-Add 3 ml of water-saturated phenol and mix.
-Add 0.6 ml of CHC l3 /Isoamylalcohol 49:1. Mix vigourously during 20 sec.
-Chill on ice for 15 min.
-Centrifuge for 20 min. in a swing-out rotor at 10 0009 at 4°C.
-Resuspend the pellet in 0.7 ml of solution D. Heating at 68°C and vortexing make the resuspension easier.
-Transfer the solution into a sterile Eppendorf tube. Precipitate 1 hour at -20°C with 1 volume of isopropanol.
-Centrifuge for 15 min. at 4°C.
-Wash with 1 ml of 70% ethanol.
-Dry the pellet at 68°C.
-Resuspend the pellet in 0.1 ml RNAase-free water at 68°C.
By using this method, one should obtain a total RNA concentration of about 1 mg/ml from 0.7 ml packed eggs.