Rated 4 of 5.
Level of importance:
Rated 4 of 5.
Level of validity:
Rated 3 of 5.
Level of completeness:
Rated 5 of 5.
Level of comprehensibility:
Rated 3 of 5.
( At the request of Raphael, I copy here the comments that I posted in my blog : https://greenfluorescentblog.wordpress.com/2015/11/18/new-data-on-smartflare-do-they-detect-mrna/ [slightly editted])
Almost exactly a year ago, I wrote a post regarding my concerns with SmartFlare, supposedly a novel method for live imaging of RNA in cells.
In a nutshell, SmartFlare are gold nanoparticles covered in oligos specific to a certain mRNA of interest. Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenchhed and “flares”, indicating the present of said mRNA.
You can read about my concerns in that older post, but apparently I wasn’t the only one concerned about their validity.
Raphaël Lévy from U. of Liverpool (UK) was concerned as well. He endeavored into this open science project to try and answer his concerns. Here ar my comments:
Raphael’s concerns were that these gold nanoparticles are maintained in endosomes and do not reach the “cytoplasm” where mRNAs reside. Since he assumes mRNAs are abent from endosomes, any fluorescent signal that we detect is an artefact.
He used several imaging approaches to test his hypothesis: electron microscopy (EM), confocal microscopy, photothermal microscopy
Unfortunately, Raphael did not perform any assesment of mRNA levels, not by RT-PCR, nor FISH. I think that for a paper that is supposed to test the mRNA detection capacity of SmartFlare, one needs to confirm that the mRNA to be detected is present in this particular system, that it responds to the treatment as published and that it does or does not co-localize with the SmartFlare. This is my main complaint about this paper, but not the only one.
Here are some of my other concerns/problems with this paper:
3. To test if SmartFlare responds to changing mRNA levels, they treated the cells with a drug which, supposedly, induces an increase of VEGF mRNA levels (VEGF is the mRNA tested here).
4. There is another option why there’s no increase in SmartFlare signal upon increase in mRNA levels – the system is already saturated. This is a matter of mathematics: on the one side, how many mRNA molecules/cell (dozens? hundreds? thousands?). On the other hand – how many particles/cell and how many oligos/particle? Do these numbers match?
5. Throughout the text, Raphael assumes that mRNA molecules are absent from endosomes. Is that proven? Since there’s a whole field of study that shows mRNAs in exosomes (extra-cellular vesicles derived from the multivesicular body), I can assume that at least some mRNAs are indeed encapsulated in vesicles inside cells. This, of course, needs to be tested on a case by case basis (VEGF in this case).
A minor point:
In the introduction (and then the discussion) Raphael mentions in vitro and in situ methods to detect mRNAs, then goes on to say how great it is if we had a system to visualize mRNA in live cells. Well, we do, more than one. We have MS2-like systems that allow live tracking of mRNAs at the single molecule level. There are also GFP-mimic RNA aptamers (Spinach, Broccoli, RNA-Mango) which enable visualization of RNA in live cells. So SmartFlare is only the 3rd option.
Overall, the only thing that was convincing, to me, was that SmartFlares are encapsulated in some kind of vesicles. I am not concinved that they do not detect mRNA inside.