Good morning Everyone. I know this might sound like a dumb question, but I can only ask at a knowledgeable community like this to get the right answer. So I intend to carry out some gene expression studies (Microarray studies and rt-PCR) in c. elegans after exposing them to a mycotoxin. Am currently at a point when I can’t say exactly what type of genes I should be studying for in the worms. Please can anyone guide me through this. The goal is to identify some genes that we believe are involved in the immune defense system of the worms. But currently the genes that I have discovered from reading the literature are only those involved in the worm’s immunity on interaction with microorganisms and not environmental toxins like am interested in. Any reference to previous studies that have studied similar compounds like mine will be highly welcome as well. Thank you
Well, qRT-PCR is only good if you know what genes to look at, right? Microarrays, passé. You should consider using RNA-seq instead. I would expect the gene expression of a ton of CYPs to be affected.
People have done work with mycotoxins like aflatoxin B1.
Thank you so much Snug. I guess you read my thoughts exactly about what am working on. I honestly really need more tips on studying about the worms using the aflatoxin.
What tips are you looking for?
The simplest experiment I can think of is to do a dose response experiment (ensure it is repeatable), figure out how high of a dosage you can push the worms and how they react to different concentrations, and do RNA-seq.
currently the genes that I have discovered from reading the literature are only those involved in the worm's immunity on interaction with microorganisms and not environmental toxins like am interested in.A fairly loose PubMed search finds 70 candidates to be this sort of paper (many of which aren't); adding the term "microarray" finds seven hits, of which six profile transcriptional response to environmental toxins. removing the term "gene expression" finds a few more, and loosening the terms in other ways finds still more. Yet more permutations might find many more hits this approach has missed, and I've only been searching using PubMed - you can do full-text searches of the C. elegans literature in much more advanced ways using Textpresso.
The term “RNA-seq” finds much less.
It looks like there are a couple dozen microarray studies of exposure to environmental toxins, and some RNA-seq, proteomic, and other profiling work - and, of course, there are still other studies of exposure to other stressors that are relevant, if only for comparison.
As a parallel approach, you might try some of these reporters induced by different types of cellular damage:
http://www.ncbi.nlm.nih.gov/pubmed/22500807 [look at some of the supplemental reporters too]
You could probably treat those different lines with aflatoxin and compare your expression pattern to those of the different RNAi inactivations in the paper. Also, do you want all of the genes with altered expression in response to aflatoxin or just a few reporters? More info on your overall goals would be helpful. Do you want to explore the transcriptional response to aflatoxin? Mechanisms of toxicity or detoxification?
Also, aflatoxins metabolites intercalate into DNA and can be alkylating agents. So you’ll likely be dealing with a DNA damage response.
All of these are really helpful forms of advice indeed. Am highly appreciative of the fact I can get such information from this forum. Thank you all.
Thank you Jordan. In response to your questions, am interested in a few reporters and mechanistic studies about the immunity developed by the animals in response to aflatoxin is my overall goal.
Then it sounds like a good starting point would be to use the panel of reporters from the Melo paper. That could provide insight as to whether specific stress pathways were involved and is less of an investment than an RNA-seq experiment would be. My feeling is that a suppressor screen for aflatoxin resistant mutants would be more informative for your stated goals than a gene expression experiment.
Thank you Jordan. But if I may ask, which of the Melo’s paper are you referring to?
This one here:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613046/ (in PMC)
http://www.cell.com/abstract/S0092-8674(12)00346-7 (directly on Cell site)
There are several reporters in the paper and a few more in the supplement that should let you look at a range of stress responses.
Thank you so much.