Filtering the cells is the
quickest way to collect them for later RNA processing. I don't recommend spinning cells
because of the chance you'll get a stress response or a temperature shock.
The following protocol is
intended for ~108Ð1010 cells. If the filtering seems slow, harvest less culture or divide
the cells over multiple filters.
If you're harvesting less than ~108 cells, you should use 25
mm filters in 2 ml tubes with a smaller filter support (09-753G, Fisher).
Plug the filter support
(K953805-0000, Fisher) in a sidearm
flask hooked to vacuum. Center a
0.45 micron nylon filter (R04SP04700, GE Osmonics) on the filter support. Place the funnel on top and carefully
clamp it all together. Start the
vacuum. Listen for any whistling
noises that may indicate a leak in the seal. If you do get whistling, make sure that the filter is
centered and free of wrinkles.
Some fraction of the filters have cracks or holes that will interfere
with the harvest. Replace the
filter with a new one if this seems to be the case.
Remove the top from a
labeled 15 ml Falcon tube. Put the
tube in a bucket of liquid nitrogen.
Once everything is ready,
remove the culture from the shaker.
Turn on the vacuum. Pour
desired amount of culture into the funnel of the filter apparatus. Watch to make sure it is filtering
properly and that no cells are making it into the flask. You can refilter if you have this
problem, but try to avoid it.
Once the culture has
completely filtered through, remove the clamp and then the funnel. Pull the vacuum hose off the
sidearm. This order is important
to keep cells from sticking to the funnel. Use forceps or a spatula to lift the edge of the filter,
avoiding the cells in the center.
Carefully roll up the filter.
Dump out the liquid nitrogen in the 15 ml tube, pop the rolled up filter
inside, loosely cap it, and dunk it back into the liquid nitrogen. Store the tubes at Ð80.
Hybrid of the DeRisi
protocol (www.microarrays.org) and a standard acid-phenol prep circulating
around the Brown/Botstein labs circa 2001
Use the small prep if
you've used 25 mm filters and the large prep for 47 mm filters.
Use RNase free reagents
and glass-/plastic-ware throughout!
Remember to use glass pipets with chloroform.
(100 ml)
2 ml |
0.5 M EDTA |
5 ml |
10% SDS |
1 ml |
1 M Tris pH 7.5 |
92 ml |
RNase-free water |
Remove a manageable set of
samples from the Ð80. They should
be in 2 ml eppendorf tubes.
Before they thaw, add 750
µl lysis buffer. Vortex, trying to
get all the cells off the membrane.
Add 750 µl acid
phenol. Vortex.
Incubate 1 hour 65C,
vortexing every 20 minutes.
Fish out the filter and
discard.
Ice 10 min.
While they are incubating,
spin the 2 ml heavy phase lock gel tubes (made by Eppendorf, sold lots of
places) for 30 sec full speed in a room temperature microcentrifuge. Set aside.
Spin lysate 5 min.
With a pipet, transfer the
top aqueous layer to the PLG tube.
Add 750 µl
chloroform. Invert to mix. Do not vortex!
Spin 5 min.
Pour aqueous layer into a
new 15 ml Falcon tube.
Add 75 µl (or 1/10 volume
if you lost some) 3 M sodium acetate.
Mix.
Add 1.5 ml (or 2 volumes)
ethanol. Mix.
Incubate Ð20C >30 min
(preferably overnight).
Spin 3000 rpm 10 min.
Wash pellet 2X with 70%
ethanol, with 2 min 3000 rpm spins between washes.
Air dry inverted on the
bench 30 min.
Dissolve pellet in 25 µl
water. You can speed up the
dissolution by heating the sample or by pipetting the pellet up and down, but I
prefer a gentler room-temperature-with-frequent-flicking approach.
Measure the concentration
with the spectrophotometer or nanodrop.
Check the quality of the
RNA on the Bioanalyzer or a gel.
Remove a manageable set of
samples from the Ð80. They should
be in 15 ml Falcon tubes.
Before they thaw, add 4 ml
lysis buffer. Vortex, trying to
get all the cells off the membrane.
Add 4 ml acid phenol. Vortex.
Incubate 1 hour 65C,
vortexing every 20 minutes.
Fish out the filter and
discard.
Ice 10 min.
While they are incubating,
spin the 15 ml heavy phase lock gel tubes (made by Eppendorf, sold lots of
places) for 2 min 1500xg in a room temperature swinging bucket centrifuge. Set aside.
Spin lysate 10 min 3000
rpm.
With a pipet, transfer the
top aqueous layer to the PLG tube.
Add 4 ml chloroform. Invert to mix. Do not vortex!
Spin 5 min 1500xg.
Add another 4 ml
chloroform to the same tube, invert to mix, and spin again.
Pour aqueous layer into a
new 15 ml Falcon tube.
Add 400 µl (or 1/10 volume
if you lost some) 3 M sodium acetate.
Mix.
Add 8 ml (or 2 volumes)
ethanol. Mix.
Incubate Ð20C >30 min
(preferably overnight).
Spin 3000 rpm 10 min.
Wash pellet 2X with 70%
ethanol, with 2 min 3000 rpm spins between washes.
Air dry inverted on the
bench 30 min.
Dissolve pellet in ~250 µl
water, adding more if necessary.
You can speed up the dissolution by heating the sample or by pipetting
the pellet up and down, but I prefer a gentler
room-temperature-with-frequent-flicking approach.
Measure the concentration
with the spectrophotometer or nanodrop.
Check the quality of the
RNA on the Bioanalyzer or a gel.