Does anyone know how to maintain large synchronous cultures of old nematodes without using either FUDR (which has recently been shown to affect the worm), or the mutant strain GE24, pha-1(e2123)? Our strain is temperature sensitive, and we need more than a thousand of day 6 worms to a perform microarray analysis. Thank you so much!!!
If you want to grow them on plates and want to use N2 as your control, unfortunately you need to do it ‘by hand’. Set up some synchronous lays/do bleaching to get your worms going and then pick to transfer.
It is quite awful, been there done that.
It’s unpleasant, but as Snug said, it’s the only way…
Is there a particular reason for using so many worms? Sufficient RNA can be obtained from 50-100 worms (which makes picking by hand a less daunting prospect).
I’ve been doing arrays on Affymetrix chips and I have to provide 13uL of RNA >100ng/uL. I’ve been using the RNeasy Microarray Tissue Mini Kit (Qiagen cat #73304) and usually my sample gives ‘Excellent’ quality on the bioanalyzer. I usually do 750-1000 worms and end up with around 27uL of 200-300ng/uL.
I would recommend on having a bit more so you can save some of the original sample and convert it to cDNA and test your results from your microarray using qRT-PCR.
Hi,
while rites of passage are often character-forming (I feel your pain Snug), are the alternatives to hand picking completely ruled out?
For example, have you considered whether cold-stress age fractionation for very old worms might be possible? Of course, I’m not sure how homogeneous your sample would be with this approach though;
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854889/
Or, if you’ve just won the lottery or have a rich lab nearby, the COPAS BIOSORT system?
Or, just for fun, seeing if the old (and slow) worms are separated from the rest of the more youthful worms when placed at one end of a large plate with a point source of OP50 at the other end? You could then chunk the oldies onto a new plate and wash them off?
Regards
Steve
We have a COPAS biosort but in awful shape since nobody has used it in a long long time. Hmm, I guess I have never thought to use it for this purpose because I do everything on plates and not in liquid culture. Worms grown in liquid culture vs. worms growing on plates are way different (as you all know) so I try to keep everything simple with less variables to consider/less hand waving etc.
as steve was suggesting u could do cold separation. it is something which i have tried and it is reasonably effective. but then
i find the cold separated worms slightly unhealthy although i dint base it on any parameters per se. its purely observational.
The best way would be to make a cold separation between L1 larvae and adults. so u wld have to to do this on a daily basis till their
reproductive cycle. but i am slightly skeptical as it involves shiftig them between ice and room temperature and then to
cultivation temperature.