Osteosarcoma lines can sometimes be difficult to transfect. Compare your transfection protocol with ours for osteosarcoma lines like MG-63 (https://altogen.com/product/mg-63-transfection-reagent-osteosarcoma-crl-1427/) and see if there are any drastic differences. Primary factors include cell confluency at the time of transfection, the procedural workflow, and the concentration of nucleic acids being transfected.

If a particular transfection reagent is going to work with a certain cell line, efficiency is usually quite sensitive to cell confluence. Also, I would test reagents of differing technologies - i.e. Fugene6/Genejuice/Effectine and not just variants of lipofectamine.

Transfections can be extremely difficult to optimize and very tough to keep consistent. There are so many variables. With lipofectamine, while confluence is a big factor, the amount of lipofectamine as well as the amount of plasmid DNA you add to the transfection cocktail can all be varied widely. Have you tried to increase the amounts of lipofectamine and/or DNA? I would try that first since the GFP expression seems pretty low.

I also agree with Michael. Have you searched the literature to see what reagent people are using to transfect cells of the same type? 

Hi Hoda,

First you did the right think about checking the transfection efficiency with a reporter gene such as GFP. One option as suggested by Michael is to try other transfection reagents and I can recommend DreamFect Gold

http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html

or even better another technology called Magnetofection such as LipoMag

http://www.ozbiosciences.com/transfection-dna/44-lipomag-magnetofection-transfection-reagent.html

You can see publications on osteosarcoma cells with these two reagents directly on the website.

Now you have to also consider that your expression cassettes of your gene of interest in not working properly; meaning that despite the good sequence, the expression is not good (bad promoter, terminator etc..). One check will be to assay the expression of your plasmid in a cell free transcription/translation assay to make sure that your mRNA and protein are expressed. This is a critical point, I believe, since you have no evidence that your plasmid carrying your gene is effectively expressed

Good Luck

Do you have the possibility, to carry out an electroporation? If not, I would also suggest, to test different transfection reagents. Maybe you could search for such a reagent at amsbio (http://www.amsbio.com/). I used one of their reagents and it worked really well (better than Lipofectamine). Right now, I am testing to transfect my cells with Lipofectamine CrisprMax, to introduce the Cas9-protein. This reagent is optimized, to introduce mature proteins.

Thank u all for your helpful answers. I've tried transfecting the cells with 5 µg DNA and 7.5 µL lipofectamine 3000 (that's the maximum amount of both components according to ThermoFisher's protocol) and it still gave nothing. We're suspecting that the sequence might've been changed during the cloning process, but it's still hard to say. 

Are you able to sequence it?

Im not personally able to do it, but I will let my superiors know. 

Additional info: I made western blotting of the *allegedly* transfected cells and they produced a giant smear, which might indicate that the protein is now over expressed.

However, my qPCR shows no change in mRNA levels of the desired protein compared to blanks. I tried changing cell line but it didn't help. 

Dear Hoda,

it is slightly unclear what exactly you transfect into the cells: protein, mRNA or plasmid expressing a protein?

Going with the plasmid option: I assume you used a standard optimised GFP plasmid for your control? If so, how exactly did you check your GFP fluorescence? If you used fluorescent microscopy how bright was the fluorescence, ie could you see it brightly straight tthrough the microscope or was it very dim and you had to use >1 second exposure to capture an image?

I'm aware of cases where people thought they had good transfection efficiency with GFP but in reality it was background fluorescence (Lipofectamine is well known to induce that in certain cells...).

mRNA levels do not necessarily equal protein levels. Did you check protein levels by WB or ELISA in addition to RT-qPCR? It could be that your mRNA is degraded quickly...

Could you post the plasmid sequence (both your intended in silico sequence and the actual sequencing data? There's a lot of things that could be wrong with the plasmid beyond the sequence of the insert, eg some expression plasmids require you to add an ATG to your gene and some already have it (in which case you need to make sure you are in frame...). Alternatively, the promoter you're using is not actually functional in your specific target cells..

Re mRNA quantification - I assume you did this by RT-qPCR? There is so many things that could be a problem in that technique ....

Best wishes,

Petra

I recommend that you get a second plasmid with your gene of interest. We recently ran into a problem where our original plasmid was expressing our gene at very low levels. Rather than try to work out the reason for that, we got a different plasmid (different vector backbone) and it expressed our gene at high levels. Both were CMV promoter driven.

If possible, test your plasmid in an easily transfectable cell line (e.g., HEK293T) to see if the problem is with your plasmid. And of course, as others have mentioned, sequence your plasmid and make certain that the cloning process was successful.

Dear Petra 

I am transfecting a GPCR in a vector backbone (pcDNA 3.1 -). I checked the transfection efficiency by fluorescent microscopy and a transfection efficiency of app. 40 % was observed 24 hour post-transfection. I checked protein levels on WB. Whenever I transfected it produced a giant smear in the lanes making it difficult to identify the band of interest. Moreover, I am unsure what it tells us - if the protein is expressed or not. And yes I investigated the mRNA levels by RT-qPCR.. 

I am not able to post the sequence as of right now as they're in y supervisor's possession. I will post them as soon as I get my hands on them. Thank u so much for your help. 

Dear Ricardo: 

The vector backbone contains both the CMV and T7 promoters. I tried transfecting in another cell line and nothing was changed - still didn't observe any difference in mRNA levels. Thank u,

Dear Hoda,

Re GPCR - most receptors are glycosylated and as such it would not be unexpected to get extra bands on the Western blot (and a smear if you have degradation of the various glycosylated forms in addition to the unglycosylated). You can de-glycosylate your sample before WB. Also, transmembrane proteins can aggregate with heating - have a look around on the web, but looks like it may work better without heating your sample. Other thing is to reduce the amount of protein loaded on WB and perhaps dilute your first antibody more. I imagine you are using an antibody that has been shown to work with your protein?

But really, it does look like you do not get expression of your GPCR from your plasmid in the first place...

If you could confirm expression by WB, then we know that your problem is with the RT-qPCR and can start trouble shooting that. However at the moment you haven't really given enough information to say anything specific...

Dear Petra 

Yes, the antibody I am using has been proven to work on my protein of interest by the supplier. 

I recently found out that the probe I am using does not detect the mRNA levels of my desired protein. So now I am pretty confident that the transfection is succesful, but I can't detect it with RT-qPCR. The WB smears must be because I load too much protein on the sds-gel. Thank u so much for your input.