Hi,
Does anyone have experience extracting RNA from a small number of worms (100 to 200)?
Specifically, how does one wash the worms free of bacteria so as not to extract bacterial RNA, yet retain all the worms without losing them during washing?
Has anyone tried sucrose flotation of small numbers of worms in an eppendorf tube?
100 worms could be picked manually after leaving the worm in bacteria -free plates
for several hours, then treat them with Trizol PTL only ( or + Column- based kit).
Sucrose flotation makes lose of worms.
Hey,
I know what Trizol is, but have not heard of Trizol PTL. What’s the PTL?
thanks
Joe
just do it as Trizol. you could not see RNAs , but proceed to end carefully and you ll get them .
When I extract RNA from a small amount of worms I usually use Glycoblue from Ambion, which supposedly increases the precipitation of small RNA amounts and at the same time make the RNA pellet more visible.
Good luck,
Marlon
Does GlycoBlue absorb in the UV spectrum? I couldn’t find this info on their web page. I can’t use it if it does. I need to measure my RNA with the spec and use equal amounts for different samples, so I can’t have any other UV absorber.
I have had pretty good success by picking a small number of worms (as few as a single worm) into 50-100 uL of water and adding the manufacturer’s recommended amount of trizol, repeated freeze/thaws, extraction, and precipitation. I use the larger volume because it is easier to work with and will decrease sample loss during extraction. I also add glycoblue to my precipitations for visibility. I usually re-suspend my RNA in a small volume and use the entire thing for RT-PCR. I let the normalization to a housekeeping gene account for small variations in RNA concentration between samples.
However, I just compared water, water + 1 uL of glycoblue, and water + 1 ul of glycogen on our spec. The readings I got after blanking with the water:
OD260 OD260/280 dilution concentration
glycoblue 0.0674 1.89 1 2.7 ng/ul
glycogen 0.0378 1.86 1 1.5 ng/ul
Thus there is a small amount of absorption at 260 that won’t alter your 260/280. Depending on how much of your sample you plan to spec it could be negligible. I usually add 0.5 uL to my samples before precipitation (half of what I measured above). If you assume the recovery is the same in all your samples it will come out in the wash anyway. Alternatively, you could try spinning your RNA sample after re-suspending it and see if you can pellet the glycogen away from your sample. Good luck.
Has anyone tried mashing the worms with a micropestle in trizol to improve yields for small worm numbers?
use the micropestle in trizol works not that easily since you can only use a small amount of trizol in the tube, and then you might run into the problem that there is too much water in your worm sample/tube (trizol can take 10% water max!).
I did get more RNA yield when leaving the worms in trizol on the vortex for 10 minutes at RT than repeated 3x freeze thawing.
The best way to isolate RNA from a small amount of worms for me is freezing the worms in trizol in a mortar (put on dry ice) and then grind them. Subsequent thawing and incubation at RT before continuing the isolation.