We have been conducting agroinfiltration experiments in cannabis plants to introduce genes of interest for studying their expression and function. Upon analyzing the results, I have noted positive signals in both DNA and RNA analyses, indicating the possible presence of the introduced exogenous genes. However, I am concerned about the potential contribution of the own bacterial DNA or RNA used in the agroinfiltration process, which might bias or even entirely account for these positive results, leading to false positives.
Are there any specific protocols or molecular analysis techniques that can help mitigate this contamination risk and ensure the reliability of results obtained in these experiments? I welcome any contributions or experiences shared on this matter.
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Well, the process of Agro infiltration itself is going to change gene expression in the short-term in your plants. Are you trying for stable or transient transformation of your plants?
There are a few things you can try to parse out bacterial vs plant contributions.
1. add some negative controls. I would suggest a buffer only control (no bacteria) that will show any changes due to the infiltration process in general. I would also suggest an Agro with an empty vector control.
2. you can use polyA-specific RT analysis for the gene expression (use oligo dT for mRNA to cDNA RT). The bacterial gene transcripts will lack the polyA tail so you won't have any showing up.
3. For DNA analysis, it really depends on what you are trying to detect. You could use Agro-specific gene primers to see if the cells are still present in your plant (they probably are, at least in the short-term).
Hope this helps and good luck!
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