At the moment, I am trying to plan a genome-wide siRNA screening in an immortalized murine macrophage cell line (J774) and would like to try as many protocols as I can before the real screen. I'll be using reverse transfection in 384-well plates. Does anyone have any recommendations? What transfection reagents have you used: hiperfect, lipofectamine, interferin, something else? How much did you need per well? If you've screened, what was final siRNA concentration that you ended up using? How was your knock-down efficiency? I'm of course doing a lot of trial-and-error titration at the moment, but it would be great to be able to hear from someone else with a bit more experience. Thanks!!
Eva,
We use a custom made protocol for siRNA transfection in 24 well-plates. We determine the amount of Interferin based on number of cells per mm^2 rather than well size alone, cell number alone or any other parameter (and that fact is of critical importance considering that all manufacturers give you protocols only based on well-plates without mentioning how many cell they seed; 50 000 cells per well in a 96 well-plate won't give the same result as 80 000 cells per well in the same format). For our protocol, 8 uL of Interferin in a total volume of 700 uL for 200 000 cells in 200 mm^2 and 10 nM siRNA leads to 80-90% KD efficiency without any cytotoxicity observed (medium is changed after 16-24h). In other words we use 0.1 uL Interferin per 2500 cells per 2.5 mm^2 (or 0.025 cm^2). To determine ideal Interferin amount this cell/surface ratio should be calculated for your experiment (for example if you use 5000 cells per 2.5 mm^2 then use 0.2 uL Interferin).
I only use primary macrophages, either BMDM or peritoneal macrophages. But according to litterature primary cell lines are known to be more difficult to transfect than immortalized cell lines (especially because they are way less tolerant to foreign DNA/RNA) so you should have no problem transfecting J774 if it works on BMDM for me, I do think you can even use a bit less Interferin than me considering that point.
Hope it helps
Alexis
send me a mail and I can give you more info on a reagent that works exceptionally well as a siRNA reagent, and also works for macrophages. I can send you a sample to test. It was developed here at KIT last year and is comparable to RNAiMax, but considerable more affordable, especially for genome-wide screens!
There are multiple publications with Accell siRNA (from Thermo Scientific) with successful use in macrophages (AM, BMDM, RAW, etc). The siRNA itself has chemical modifications which facilitate passive uptake by cells.No transfection reagents are required. Here are the results I found today (search terms "Accell" and "J774", 4 of the articles are free). In one case, Accell siRNA was used when amaxa nucleofection was not tolerated by the cells (alvelolar macrophages).
http://highwire.stanford.edu/cgi/searchresults?fulltext=accell+J774&andorexactfulltext=and&searchsubmit=redo&resourcetype=1&x=51&y=13
Good Luck!
We use Interferin with a concentration of 10 nM of siRNA, works well with a KD efficiency of up to 90% in human as well as murine macrophages.
Hi Everyone,
Thanks for your advice! Gary, your transfection reagent sounds great. I'll be sending you a mail in a minute. Alexis,what amount of interferin do use per well, and what kind of murine macrophages are you using? Are they immortalized cell lines? Do you have any experience with J774s? (Sorry for answering your answer with more questions, and thanks so much for your help!)
Best,
Eva
Eva,
We use a custom made protocol for siRNA transfection in 24 well-plates. We determine the amount of Interferin based on number of cells per mm^2 rather than well size alone, cell number alone or any other parameter (and that fact is of critical importance considering that all manufacturers give you protocols only based on well-plates without mentioning how many cell they seed; 50 000 cells per well in a 96 well-plate won't give the same result as 80 000 cells per well in the same format). For our protocol, 8 uL of Interferin in a total volume of 700 uL for 200 000 cells in 200 mm^2 and 10 nM siRNA leads to 80-90% KD efficiency without any cytotoxicity observed (medium is changed after 16-24h). In other words we use 0.1 uL Interferin per 2500 cells per 2.5 mm^2 (or 0.025 cm^2). To determine ideal Interferin amount this cell/surface ratio should be calculated for your experiment (for example if you use 5000 cells per 2.5 mm^2 then use 0.2 uL Interferin).
I only use primary macrophages, either BMDM or peritoneal macrophages. But according to litterature primary cell lines are known to be more difficult to transfect than immortalized cell lines (especially because they are way less tolerant to foreign DNA/RNA) so you should have no problem transfecting J774 if it works on BMDM for me, I do think you can even use a bit less Interferin than me considering that point.
Hope it helps
Alexis
Hi Alexis,
Yes, that helps a great deal, and, you're right: I'd completely forgotten to ask you about cell density. I'll try your protocol as soon as my interferin sample arrives. Thanks!
I had great results on J774A.1 by using RNAiMAX lipofectamine from invitrogen (2uL/well in a 6-well setting) and siRNA from Ambion (25pmol/well). I observed negligible toxicity and a silencing efficiency ranking from 5 to 10 fold 48h after transfection, depending on the gene.
Hope this helps
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