I'm wondering if someone used or is going to use nanoflares (smartflares) to isolate cells based on target mRNA sequences.
I'm trying to do it but still have some problems.
Could anyone help me?
I've had experience with this technology. The major problem I had is that uptake of the nanoflare particles tends to vary from cell to cell. So a cell may come up as negative for that mRNA using smartflare, but it doesn't actually mean that it does not express the transcript of interest. For example, if I FACS sort cell based on nanoflare-targeted mRNA expression and do a qPCR to quantitate enrichment of that transcript in the "positive" population relative to the flow-through, I do not see any enrichment of my transcript of interest.
What are the problems you are experiencing?
Yes, variable cell up-take of the nano gold is a major problem. You should always use a nanoflare for 18S in parallel with your gene of interest as a positive control.
I used primary cells so tech support told me to try first uptake control plus scramble. But when i analized my cells for uptake i had fluorescence of scramble comparable of uptake and more of non treated cells. So i asked to tech support " is the scramble control broken or maybe the sequence is linked in aspecifically way?" They answer me "it could be the second one" but nothing else! So what i need to do? My cells have also a good uptake. I'm buying the assay for my target gene and one housekeeping control so i could check maybe better my fluorescence signal. Or at least the tech support suggest me.
The ease with which you can do this will depend on the level of mRNA expression. The scramble control gives you an idea of the background level of signal produced by the probe being spontaneously released. This suggests that there is a certain degree of background noise. The expression of your gene of interest would have to get above this noise level to be useful for sorting.
I Agree with you. Thank you for all the answers. I will let you know about the next step with target gene.
Hi Mariavittoria, Ali, Kenneth, Martin
If you are still interested by the SmartFlares, you might be interested by our blog post, open science notebook, and open data repository detailing our experience with these particles, as well as the many comments from various experts on the blog post. The conclusion is that the SmartFlares remain in endosomal compartments and do not detect mRNA levels. The increase in fluorescence is due to nucleases. (we have also observed the cell-to-cell variability). We are writing this up and it should be formally published soon.
If you have done further work with SmartFlares, I would love to hear more from you, and of course feel free to leave a comment at the blog.
https://raphazlab.wordpress.com/tag/smartflares/
https://raphazlabcommons.wordpress.com/tag/smartflare/
http://cci02.liv.ac.uk/gallery/show_project/1217/
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