I am new in the field of siRNA since my main field is Pharmaceutical Technology.
My research interest recently focused on the possibility of preparing a lipidic or polymeric carrier to ensure the stability of siRNAs and their transfection in neuronal cells, with the possibility of a future in-vivo use of such carriers.
I have some questions about handling of siRNA:
1) I read in several online forums and in various protocols many different suggestions about how to treat glassware, surfaces and plasticware: since I don't have the possibility of baking my glassware and I already have a lot of non-sterile plasticware, do you think is effective to soak all these items in DEPC-water overnight, then autoclave everything for few hours to eliminate the DEPC? For how long do you think I can use these items if I store them in sealed plastic bags?
2) Do you think that RNAse can be present in chemicals? I will use phospholipids, cholesterol, peptides (from chemical synthesis), and Sephadex G 50 to run a chromatography column: do you suggest to treat these items in some way before putting in contact with the siRNA?
3) Can syringes for human use be considered RNAse-free (since they are sterile)?
4) Do you suggest to work under a laminar flow hood or it is enough to pay attention to air currents, people around, using gloves and deeply cleaning every surface?
I already read some advices about handling conditions for RNA extraction from cells, but I would like to know if conditions have be the same in my case, since the siRNA duplex (19 nt base pairs + 2 dT overhangs) with phosphorothioate modifications should be more stable against rnases.
Dear Michele,
your research it's quite interesting...keep us update on how is it going!
on your request:
1-soaking in DEPC-treated water could work...but not already treated water, but use DEPC directly, in water overnight in agitation then autoclave. Storing in sealed plastic bag could help, remember that if you open it more than 2-3 times you have to redo the treatment
2-RNAse is everywhere! apart from jokes...the only two critical items (for me) could be peptides and sephadex...for peptides, diluite them in DEPC-treated buffer at least you lower the RNAse content..for sephadex, make a pre-run in DEPC-water, again to diluite rnase
3- if not explicit stated they are not RNA-se free...wash also in DEPC-treated water
4-better safe than sorry...work under laminar flood
and yes it is true that siRNA duplex are a little more stable than normal RNA..but again..better safe than sorry :)
good luck!
Hi Michele,
Shortly:
1 - Your method seems a good idea to me. Similar treatment is applied to all materials when you are performing mRNA in situ hybridization and it is very effective. Materials should be used in a week time maximum.
2 - Could you treat your Phospolipids, cholesterol, peptides with UV light (at least 20 min)? I guess it will depend on stability of those under those conditions.
Sephadex G, I would wash the column and HPLC system for a good while with DEPC-treated water.
3 - Sterile does NOT mean RNase free. For my experiments I have always used RNase free tips and eppendorfs to prepare siRNA nanoparticles. But, honestly I think there is no reason to be so worried about this issue since you will most likely transfect your cells in serum-containing media. As you know serum is full with RNases... and in the end is actually a good way of testing if your formulation protects siRNAs from endonuclease degradation.
4 - I think similar care to what you use for RNA extraction may suffice, however remember if you are applying those siRNA nanoparticles onto cells you need to work on a "sterile" environment, such as a tissue culture.
Best of luck
https://www.thermofisher.com/us/en/home/life-science/rnai/synthetic-rnai-analysis.html
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