Worm Breeder's Gazette 14(3): 10 (June 1, 1996)
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.
| 1 | Department of Cell Biology, Yale University School of Medicine, New Haven CT. 06520 |
| 2 | Departments of Genetics, Yale University School of Medicine, New Haven CT. 06520 |
The following protocol uses TRIZOL reagent from Gibco-BRL to isolate RNA
from whole worms. TRIZOL reagent is a solution of guanidine
isothiocyanate and phenol which simplifies the original method published
by Chomczynski and Sacchi1 and can be used to isolate RNA, DNA and
proteins from the same sample. The following protocol does not involve
sonication and results in good yields of RNA which have been used in our
laboratory for Northern analysis, RT-PCR, RACE reactions, and cDNA
synthesis.
For 2mls of worms grown in liquid culture:
1. Add 8mls of TRIZOL reagent to 2mls of packed worms in a 15ml
polypropylene tube.
Vortex and invert tube to solubilize and lyse worms. {NOTE: Getting
the packed worm pellet into solution can take some time and vigorous
vortexing. This is fine because the longer you let the worms sit in the
TRIZOL, the better the lysis and thus more RNA will be isolated. Letting
the solubilized worm pellet sit in TRIZOL for at least five additional
minutes is recommended. At this step the solution should be pink and
will contain thread-like insoluble material.}
2. Split solution into 8 epi-tubes. About 1250ul per tube.
3. Incubate at RT for additional 5 min. Spin 14K for 10min at 4oC in a
microfuge to remove insoluble material.
4. Remove liquid to a fresh epi-tube. Add 200ul CHCl3 to each tube.
5. Invert/Vortex for 15sec. Let incubate at RT for 2-3min. {NOTE: I
found with the guanidine isolation that the 15sec vortex is critical.
Too little vortexing at this point can result in "smeary" RNA preps. I
have no explanation for this.}
6. Spin 15 min. at 14K at 4oC to separate phases.
7. Remove upper aqueous phase to a fresh tube. Add 500ul isopropanol
and mix.
8. Incubate 10 min. at RT to precipitate RNA. Recover by spinning at
14K for 10 min. at 4oC.
9. Carefully remove aqueous away from pellet. {NOTE:Pellet will be very
white.}
10. Wash pellet with 100ul of 75% EtOH in diethyl pyrocarbonate (DEPC)
treated H2O. Vortex briefly. {NOTE:Pellet will often float free. Also
RNA pellets can be stored in the 75% EtOH at -80oC for up to one year
safely.}
11. Spin at 7.5K for 5 min. at 4oC.
12. Remove supernatant and air dry pellets for 5-10 min.
13. Vacuum dry pellet for ~7 min. WITHOUT centrifugation. {NOTE:
Extensively drying RNA pellets makes them hard to resuspend.}
14. Dissolve pellets in 25-50ul DEPC-H2O. To help dissolve heat at
60oC for 10 min. {NOTE: If RNA won't resuspend completely you may have a
good yield and more DEPC-H2O can be added. I have often had to add from
200ul to 500ul to get a good resuspension.
15. Dilute 1-5ul of prep into 1ml of dH2O and take OD 260 & 280 to
determine concentration and purity. {NOTE: An A260/280 ratio of <1.6
indicates partially dissolved RNA.}
My yields have typically been between 1-4mg of total RNA from 2mls of
whole worms.
I would like to thank Jeff Yuan for "field testing" the protocol.
1. Chomczynski, P. and Sacchi, N. Anal. Biochem 162, 156 (1987)