I have a the problem that has been bothering me for 7 months in the lab. My thesis focuses on the study of a transcription factor in the movements of gastrulation (EMT) and I work with explants of xenopus cap explants as a model. My problem is the unproducibility of the results. When I inject mRNA in the embryo, The QPCR analysis of the expression of certain genes in the animal cap assay does not give me the results expected and when I repeact the experiment the results are differents . I have tried several times to optimize my work method but so far I do not know where is the problem . can anyone explain me who I can improve to get reproductible result with ? I am open to all your advices , I am really desapointed by these results.
thank you
Dear Arnaud,
I would like to help you but I need some informations.
could you be more precise in the following points:
- At which stage you inject? 2-4 or 8 cell stage?
- Specify nl you inject in EACH blastomere (20-10-5nl?), also pg/ng of capped mRNA for EACH injection.
- Do you select your well injected cap (using GFP as a tracer for exemple)?
- What medium you use to growth your caps (MBS, MMR, NAM)? Specify also the concentration (0.1x, 1x).
- You make explants at blastula (of course), at which stage you collect caps? before (st10.5-to-13/14) or after neurula stege (st15-to-20)?
- Are your cap growth in a plastic or glass dish? Do you use agarose?
- Your TF works in EMT (mesoderm, so it should be required at the beginnning of gastrulation st.10, i guess), can your TF convert cap (which will became ectoderm/multiciliated epithelium) to mesoderm? Do you observe dissociation?
-The name of your TF of interest is not required, but if you are working on a TF involved in Wnt/non canonical, it will be a bit tricky (mRNA dose will be really really, again really, important).
If you are a PhD student, 7months is 1/5 of your time....I hope I could help you, so in 3/4 weeks you should obtain your result (1 week for one experiment, you should reproduce your experiment 3/4 times = 1 months).
Allez, courage!
Pierluigi
ps: check mat/met in my paper here
Article Lineage commitment of embryonic cells involves MEK1-dependen...
pps: Eric Bellefroid's Lab, j'imagine.
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