Compared to cells like HeLa or 293T, how easy is it to transfect DNA and siRNA into A549 cells? Does it require different/special reagents?
transfect 1ug EGFP plasmid into one well of 6 well plate of A549 cells by lipofectamine 2000 (2ul) , more than 80% cells turned green 24 hours post transfection.
I do not think it will be more difficult than HepG2 cells, try interferin reagent and do reverse transfection technique. Trypsininzed cells over siRNA. Should work good.
Cheers
It seems to be pretty common. Here are three examples including one siRNA transfection. I strongly suggest you to contact the authors for more details on the protocols. These are free full text papers on PMC.
Good luck!
J Exp Clin Cancer Res. 2011; 30(1): 20. Published online 2011 February 17. doi: 10.1186/1756-9966-30-20 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3051912/
"A549 cells were seeded into 6-well plates and transfected with the miR-415-expressing vector or the control vector expressing a non-specific miR-NC using Lipofectamine 2000 (Invitrogen), and were selected with spectinomycin (100 μg/ml) to generate two stable monoclonal cell lines (a miR-218 stable cell line, A549/miR-451, and a control stable cell line, A549/miR-NC)."
BMC Immunol. 2011; 12: 16.
Published online 2011 February 17. doi: 10.1186/1471-2172-12-16 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048559/
"The human lung carcinoma A549 cells were obtained from American type culture collection (ATCC) (Manassas, VA, USA) and were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO-BRL, Carlsbad, California, USA) at 37°C in a 5% CO2 incubator. A total of 1 × 106 cells were grown to 70% confluence in 100 mm2 culture plates before transfection. The transfection reaction was performed by using Lipofectamine plus reagents (Invitrogen, California, USA) with 2 μg of each plasmid, pEGFP-C3, pEGFP-3CLpro, and pEGFP-C145A according to the manufacturer's instructions. The cells were then cultured in serum-free DMEM for 12 hrs at 37°C in a 5% CO2 incubator and subsequently in DMEM with 10% FBS."
J Exp Clin Cancer Res. 2011; 30(1): 63.
Published online 2011 May 28. doi: 10.1186/1756-9966-30-63 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3118969/
"The 25 nM, 50 nM and 100 nM siRNAs were transfected into culture cells with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA), according to the manufacturer's protocol. The cells were harvested 24, 48, or 72 h after transfection for analyses. Also as controls, A549 cells were either untreated or treated only with Lipofectamine 2000 reagent."
I have recently transfected A549 using GeneJuice at a pDNA dose of 200 ng pr well and RNAiMAX at a concentration of 30 nM. Both resulted in efficient transfection. However, if I reduced the dose of pDNA/siRNA the efficiency decreased rapidly. Further increase did not improve the efficiency.
FuGene HD and Lipofectamine 2000 should also work as I have seen them being used in several papers...
96 well, the siRNA volume was 50 uL. Weight (pDNA) to volume (GJ) ratio 1:3 for genejuice and 13.3 uL RNAiMAX/ug siRNA.
It´s very easy. Invitrogen have protocols to how to transfect all cells, including A549. At least on these protocols have the plates and volume to applicate.
transfect 1ug EGFP plasmid into one well of 6 well plate of A549 cells by lipofectamine 2000 (2ul) , more than 80% cells turned green 24 hours post transfection.
A549 should be sufficiently simple to transfect, so long as you have the right reagents for it. See this one, which I believe has attached cell markers that make it specific to A549 cells.
https://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/
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