Worm Breeder's Gazette 10(2): 154

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Rapid Small Scale Worm RNA Preps

Dave Pilgrim

Borrowing yet again from yeast molecular biology, the following 
procedure has been used to isolate clean, intact RNA from small 
quantities of worms.  This saves the time and effort of growing up, 
and grinding in liquid nitrogen, large liquid cultures.  This 
procedure should make it possible to process easily 10 to 20 stocks in 
parallel for screening by Northerns.  Yields are ~200 g of clean RNA 
from 4x9 cm 'high yield' plates (see below), or about 2 g of clean RNA 
from a single, 5 cm streaked plate.
Mini-prep.  Wash worms off 4 plates (5 cm) that are just clearing.  
If you want, the worms can be incubated in S-media for 30 min to 
digest whatever they have in their gut, and then purified by standard 
sucrose flotation in a 5ml tube.  Resuspend the worm pellet in 0.5 ml 
of liquid.  (Use any RNAase-free, solvent resistant, sterile tube that 
is big enough.  I use Falcon 2098 50ml disposable polypropylene tubes.)
Add 2ml GuEST buffer and 2ml PCI (see below), and then 6g glass beads.
Vortex on high speed, using a regular bench vortex, for 2 min at 
room temp.  Draw the liquid off the beads with a pipetman into 4x1.5 
ml eppendorf tubes, rinse the beads with 0.5ml of PCI, and add this to 
the microfuge tubes.  Microfuge for 2 min.  Transfer the aqueous layer 
to fresh tubes, add 1/10 vol 3M NaAcetate, pH 6.0, and re-extract with 
PCI.  Spin, transfer the aqueous phase (leaving the interface, if any, 
behind), and precipitate the RNA by the addition of 2 vol of 100% 
ethanol.  -20 C for 20 min, spin in microfuge 10 min, decant, and wash 
pellet with 80% ethanol.  Dry, and dissolve the pellet in 0.3ml of 
dddH20 (see below).  Add 0.9ml of 4M NaAcetate (diethylpyrocarbonate (
depc) treated), and let sit at least 5 hr at 4 C.  Spin in microfuge 
10 min, discard supernatant.  Dissolve the pellet in 0.1ml dddH2O, add 
5 l 3M NaAcetate, and 210 l 100% ethanol.  Precipitate at -20 C for 20 
min, spin, wash with 80%, and dissolve the final pellet in 50 l dddH2O.
Dilute 2 l to 1ml with dddH20, read A260 and multiply the absorbance 
reading by 20 to get the RNA concentration in mg/ml.  p(A)+ selection, 
using poly(U) Sepharose (Jacobson [1987] Meth.  Enz.  152:254261), 
should give 2-4 g of p(A)+ RNA, although using total RNA has given me 
good signals on a Northern with an actin probe.  If you are in a hurry,
you can skip the NaAcetate precipitation step, since large MW DNA 
won't transfer upon Northern Blotting.
Micro-prep.  Wash the worms off a single 5 cm plate that is just 
clearing.  Repeat the above procedure, using 50 l worms, 100 l GuEST 
buffer, 100 l PCI and 0.5g glass beads.  Rinse beads with 50 l PCI.  
Do the salt precipitation of RNA in 30 l by adding 90 l of 4M Na 
Acetate, and dissolve the final pellet in 10 l.
Frozen Worms.  I have used this procedure, successfully, to prepare 
poly(A)+ RNA from frozen worms.  Washed worms were suspended in 0.1M 
NaCl, quick-frozen in liquid nitrogen, and kept at -70 C for 1 month.  
Use 4ml of GuEST, 4ml of PCI and 12g of beads per ml of worms.  Thaw 
the tube just until the frozen plug of worms can be removed, and drop 
the plug into the GuEST/PCI.  Proceed as with the mini-prep, although 
you may want to vortex a bit longer.
Buffers.  GuEST (From M.  Goedert) Add 245ml sterile distilled water 
to 200g Guanidine isothiocyanate (BRL Ultra Pure).  Add 21ml 1M Tris 
pH 7.4, and 42ml 100mM EDTA.  Heat gently to dissolve.  Add 9ml 
Sarkosyl, and 4.2ml  -mercaptoethanol.  Bring volume to 420ml with 
sterile distilled water.  Filter through a sterile Nalgene filter, and 
store at 4 C.
PCI.  Phenol:Chloroform:Isoamyl alcohol, 
Glass Beads.  0.3 to 0.4 mm diam (although I haven't tried other 
sizes).  I borrowed mine from J.  Kilmartin, but apparently BDH and 
Sigma sell them.  They can be acid washed, baked and re-used.  dddH20. 
Add depc (0.07% v/v) to sterile, double-distilled water, shake for 10 
min, and then autoclave.
'High Yield' plates.  (From A.  Spence) Add a drop of 20% glucose to 
a large NGM plate, and then spread the plate with a wild-type bacteria 
such as NA22.  Let sit overnight before adding worms.
HINTS: Wear gloves, use only sterile tubes and pipette tips, and 
generally treat the RNA as you would HIV, and you should have no 
problems.  A quick method for checking the quality of your RNA is on a 
0.8% agarose minigel.  Denature the RNA for 10 min at 65 C, chill on 
ice and add sterile glycerol/dye/buffer.  Run 2 g RNA/lane in a RNAase 
free gel box with standard gel buffer, ~7 V/cm for 40 min.  EtBr stain 
5', photograph.  You should see two tight, rRNA bands, with no high MW 
DNA visible.  Avoid using old plates, where the worms have started 
burrowing, as the softened agar tends to wash off with the worms, and 
contaminate the RNA.  In these cases a small-scale sucrose flotation 
might be useful to clean up the worms, but I haven't yet tried this.