For gene expression/ transcriptome analysis, which stage of the life cycle of the worm C. elegans (L1-L4 or adult) is most suitable for isolation of good quality RNA?
If RNA isolation is done from adult worms, what age of worms (i.e. how many days post-L4) will be most suitable.
What minimum population size of worms is likely to be sufficient for getting enough amount and quality of RNA?
Dear Vijay Kothari
For gene expression/ transcriptome analysis, which stage of the life cycle of the worm C. elegans (L1-L4 or adult) is most suitable for isolation of good quality RNA?
The L4 stage would be the most appropriate stage because at this stage the L4 worms are actively growing and also show significant cellular changes.
If RNA isolation is done from adult worms, what age of worms (i.e. how many days post-L4) will be most suitable.
I would not recommend RNA isolation from adult worms primarily for two reasons, 1) the presence of RNases and 2) the tough cuticle that makes it difficult to access the cellular contents, including the RNA, without proper disruption. This requires specialized methods, such as repeated freeze-thaw cycles or enzymatic digestion, which can potentially lead to RNA degradation. So, I would not prefer adult worms.
What minimum population size of worms is likely to be sufficient for getting enough amount and quality of RNA?
To get sufficient amount of RNA, you will have to use between 2000 to 8000 worms, but this will depend on the specific method used and desired RNA yield. The amount of RNA obtained per worm will vary, and studies have reported around 19-21 ng per adult worm.
Best,
It's going to depend on your question and if the presence of gametes/embryos is going to "complicate" your experiment.
Are you looking for genes that are expressed in response to a stress? during normal growth & development? etc.
L1 is nice because it has an exact # of cells and no gametes yet. Easy to bleach to synchronize & can get 1000s of worms all the same age.
Adult is the easiest to get lots of "raw material" since they are so large, but the gametes & embryos have their own gene expression patterns.
Either way, you'll want to both physically grind to disrupt and use proteinaseK to break open the cuticle.
Good luck!
Vijay Kothari The adult stage of C. elegans is generally the most amenable for RNA extraction. Adults provide a larger biomass than earlier stages like L1–L4 larvae, yielding more total RNA. Additionally, adult worms are easier to collect and handle in sufficient numbers for consistent RNA extraction, making them ideal for most gene expression studies. However, if stage-specific expression is important, synchronized populations at the desired developmental stage may be used, though RNA yield will typically be lower in earlier stages.
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