I am using RAW264.7 cell line and tray to transfect with different concentration of micro-RNA (mimic & inhibitor) by using lipofectamin 2000 as a transfection reagent and check the result by qRT-PCR but have no diffrence. I also use some mico-RNA labeled with fluorescence substance but still the transfection is failed, i also change the transfection reagent . the medium I am using for cell culture is DMEM and try both with FBS and without FBS with 60-80% of cell confluencey (all reagents are prepared according to the company protocol).
As it has been mentioned several times her on ResearchGate, transfection of macrophages with chemical approaches is not reasonable as either macrophages got activated and apoptotic or accumulate the transfection reagent in endosomal compartments resulting in unwanted side-effects. We are currently preparing a manuscript on the systematic evaluation of commercially available kits that will demonstrate that almost all methods are not suitable.
From my point of view, the only suitable method is a modified Nucleofection approach that has been developed and optimized by my group. We have not yet tried to transfect miRNA but I expect that the methods will also work with miRNA similarly to the siRNA approach we have developed. However, we have not yet tried to transfect RAW264.7 macrophages. Please see the following publications for more details:
Scherer O, Maeß MB, Lindner S, Garscha U, Weinigel C, Rummler S, Werz O, Lorkowski S. A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection. J Immunol Methods 2015; 422:118-24.
Maeß MB, Keller AA, Rennert K, Mosig A, Lorkowski S. Optimization of the transfection of human THP-1 macrophages by application of Nunc UpCell technology. Anal Biochem 2015; 479:40-2.
Maeß MB, Wittig B, Lorkowski S. Highly efficient transfection of human THP-1 macrophages by nucleofection. J Vis Exp 2014; (91):e51960.
Maeß MB, Wittig B, Cignarella A, Lorkowski S. Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli. J Immunol Methods 2014; 402(1-2):76-81.
Maess MB, Buers I, Robenek H, Lorkowski S. Improved protocol for efficient nonviral transfection of premature THP-1 macrophages. Cold Spring Harb Protoc 2011; 2011(5):pdb.prot5612.
Schnoor M, Buers I, Sietmann A, Brodde MF, Hofnagel O, Robenek H, Lorkowski S. Efficient non-viral transfection of THP-1 cells. J Immunol Methods 2009; 344(2):109-15.
Have you used Lipofectamine RNAiMAX because I used this reagent and got very good up-regulations? I detected depending on the transfected miRNA in increase of 4 to 500 fold by performing a reverse transfection but you cell are suspension cells or?
What detection method you are using for your miRNA? I used the stem-loop reverse transcription published Caifu Chen 2004 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1292995/
and modified by Varkony-Gasic
http://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-3-12
Maybe you have to test several other reagents who cause a good transfection in your macrophages because in certain cell types you have to take a special transfection reagent.
As it has been mentioned several times her on ResearchGate, transfection of macrophages with chemical approaches is not reasonable as either macrophages got activated and apoptotic or accumulate the transfection reagent in endosomal compartments resulting in unwanted side-effects. We are currently preparing a manuscript on the systematic evaluation of commercially available kits that will demonstrate that almost all methods are not suitable.
From my point of view, the only suitable method is a modified Nucleofection approach that has been developed and optimized by my group. We have not yet tried to transfect miRNA but I expect that the methods will also work with miRNA similarly to the siRNA approach we have developed. However, we have not yet tried to transfect RAW264.7 macrophages. Please see the following publications for more details:
Scherer O, Maeß MB, Lindner S, Garscha U, Weinigel C, Rummler S, Werz O, Lorkowski S. A procedure for efficient non-viral siRNA transfection of primary human monocytes using nucleofection. J Immunol Methods 2015; 422:118-24.
Maeß MB, Keller AA, Rennert K, Mosig A, Lorkowski S. Optimization of the transfection of human THP-1 macrophages by application of Nunc UpCell technology. Anal Biochem 2015; 479:40-2.
Maeß MB, Wittig B, Lorkowski S. Highly efficient transfection of human THP-1 macrophages by nucleofection. J Vis Exp 2014; (91):e51960.
Maeß MB, Wittig B, Cignarella A, Lorkowski S. Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli. J Immunol Methods 2014; 402(1-2):76-81.
Maess MB, Buers I, Robenek H, Lorkowski S. Improved protocol for efficient nonviral transfection of premature THP-1 macrophages. Cold Spring Harb Protoc 2011; 2011(5):pdb.prot5612.
Schnoor M, Buers I, Sietmann A, Brodde MF, Hofnagel O, Robenek H, Lorkowski S. Efficient non-viral transfection of THP-1 cells. J Immunol Methods 2009; 344(2):109-15.
Hi Steffan and Tariq (and followers)!
Very interesting comment from Steffan. I totally agree that chemical transfection can be hard for macrophages but I will limit this to plasmid transfection. As mentioned, most of mononuclear phagocytes (incl monocytes, macrophages and DCs) are "resistant" to plasmid transfection simply because they have a battery of self-defense mechanisms (e.g. activation of AIM2/IL or cGAS/IF cascades in response to foreign DNA).
A great alternative is to transfect mRNA instead (if possible modified mRNA) and the problem is solved.
Regarding RAW macrophages, I would like to add that
- siRNA or miRNA transfections are not at all so difficult (you can easily reach 80-100% of KD by transfecting siRNA into these cells with Viromer BLUE or GREEN)
- transfection of pDNA is achievable with Viromer RED (50-60% of transfected cells), even if mRNA is more efficient (up to 80%).
Hope it helps, please ask me if you need more info.
Bests, Sandra
Recent technology but already some publications...
siRNA / miRNA TRANSFECTION INTO RAW 264.7 MACROPHAGES:
An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.
Robertson et. al., PLoS Biol, 2016
MicroRNA-29b modulates innate and antigen-specific immune responses in mouse models of autoimmunity.
Salama et. al., PLoS One, 2014
PLASMID TRANSFECTION INTO RAW 264.7 MACROPHAGES:
Delta-Like Ligand 4 Modulates Liver Damage by Down-Regulating Chemokine Expression.
Shen et. al., Am J Pathol., 2016
- Try the Accell Delivery Media (Cat #B-005000)
https://dharmacon.horizondiscovery.com/rnai/ancillary-reagents/accell-sirna-delivery-media/
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