RNA isolation of L1

Model Organism: Worms Methods & Reagents
1

Hallo all together,

i’ve tried to isolate RNA from L1 worms but failed . I use Trizol/chloroform in combination with an RNA-purification kit but i also tried only Trizol.
For RNA isolation i used 1000 and 2000 worms but no RNA was detectable. How many worms do you use for RNA isolation of L1?

Thanks a lot!

2

Hi,

leaving aside the method for one moment, one can calculate (very roughly) how much RNA one might expect to get from 1000 L1 worms.

I refer you to the new and refreshingly informative board ‘Facts & figures’;

http://forums.wormbase.org/index.php?board=334.0

If we assume that an adult worm has approx. 20ng of RNA in a total mass of ~3.2µg per worm and we use the same ratio (which is probably wrong and can be corrected in a good spirited way by someone) for an L1 worm with a mass of ~0.13µg (240µm x 25µm), then each L1 worm has ~0.8ng RNA.

hence 1000 L1 worms makes 800ng of RNA. Using Ribogreen or similar and a plate reader (and not a standard 260nm/280nm spec) one can detect pg quantities of RNA (once the DNA has been DNased away). So I would take it that you should be able to detect RNA from your prep.

As for extraction methods, there are many already in the literature and also the Wormbook.

see also:
http://forums.wormbase.org/index.php?topic=979.msg1891#msg1891

Steve

3

I just finished the better part of 6 months fighting this same question, so here the wisdom of that experience.
I tried every variation of trizol extraction methods used in worms I could find. no amount of incubating/vortexing/freeze cracking
ever worked for L1s. The only methods that I got reliable non degraded RNA from was either grinding in a dounce homogenizer in trizol or
grinding in a mortar and pestle in liquid nitrogen and dissolving the worm powder in trizol. In either case, you need to do the chloroform
extraction to get rid of the worm debris.

I ended up using close to 2500 worms (to account for loss) per extraction
and I averaged around 1.8µg RNA from that, which is about .72ng per worm.

4

Thank you very much!
How much trizol did you use, when grinding in a dounce homogenizer, Nicoli?

kind regards

5

I used qiagen’s recommended amount for animal tissue which is 700ul . Make sure you are removing as much of the supernatant
from the worm pellet as possible, there is a limit to how much water trizol can handle.

The only step you want to work quickly at is moving the worm pellet in trizol to the dounce. Once they have been broken apart
the RNA is supposedly protected in trizol, so to minimize loss I would pull the handle out 3/4 from the vessel and let the trizol-worm slurry on the sides/handle
collect at the bottom for a couple of minutes before removing it for the rest of the protocol.

Anyways I hope this saves you from the frustration I had. Good Luck!

6

Thanks again for your expertise. The tip with the grinding in trizol work out. Out of 2500 L1 i got enough RNA :slight_smile: !