Hi Gianluca, if I would be you, I would prefer the stable transfection with an inducible construct, expressing shRNA.

Hi, Gianluca, I think bot options will be suitable. Transient transfection you can use as a preliminary (first) evidence, that after transfection you have a alteration in suppressor expression (with qPCR of WB, if you have antibody). However, you still require stable transformation as well, better, as Daria mention, with inducible construct. Good luck!

Both of them are lots of work at the beginning, of course. Then let's do the stable transfection!! :)

siRNA is transient so you may not see the effects of knockdown in 48 - 72 hrs. Do you know the half-life of your protein? If it is stable and you get a 80% knockdown of RNA (using siRNA) your protein may still be present so you may see no effect. I would try shRNA and even cloning and transfecting the gene into cell lines to see what effect that has on cells.

Dear Gianluca,

I would first try to find out using transient transfections and qRT-PCR which siRNA is inhibiting you mRNA best. (out of 5 only 1 was very efficient, 1 medium eff and 3 did not inhibit) This can be done in any cell line, as long as it expresses your mRNA of interrest..

The fact that the transfection efficiency of your cells is only 20%, most probably makes it difficult to appreaciate the effect. You could work at the transfection efficiency to get it much higher,but most probably stable transfection is best. Keep in mind that your siRNA is already active if you do not use a inducible system. We silenced our gene by creating a lentivirus, with which we could transduce our cells for approx 90%.. This worked perfect.

It depends on your experiment. If you need to follow the silenced cells for more then a few days then stable transfection is defiantly the way since you would lose silencing if you rely on transient transfection as you lose plasmids when cells divide. The suggestion of using an inducible system is good although I didn't have much luck with inducible shRNA in the past when using pol3 promoter based plasmids but that should be solved if you use one of the miRNA like expression vectors that are expressed of a pol2 promoter which open all the available inducible systems.

I also opt for sh constructs. You have Stefano Indraccolo in Padua who is an expert and a really kind person, ask him!

Thanks to all, for the interesting answers.

Aiming to evaluate the role of our gene in tumoral transformation, as some of you suggested I plan in parallel to perform overexpression experiment.

Regarding the silencing, my idea is to perform a series of different experiments in order to test the effects on cell viability/cell growth including colony formation assay. I haven't considered the use of an inducibile system but sounds it could give a more reliable model to work with. I plan to buy shRNA Plasmids, do you have experiences with some companies?

Hello Gianluca:

Well, first I'd say I totally agree with Mark Sutherland, the whole process really depends on your gene product, the protein, in terms of stability, half-life, etc. Since you do plan to perform functional analysis (and probably don't care about gene expression per se), you should first have above information.

I am not sure if your cell lines are suspending (not adherent) in which case the transfection (siRNA) will be definitely difficult and the efficiency low (based on my previous experiences with malignant plasma cells, even with multiple transfections). I have tried several transfection reagents, but I think the more optimistic approach is trying shRNA using a more effective transfection tool (if you think knocking down the gene will answer your question).

I would try both, but also check the protein expression in transfed cells. In additional, I would also include a functional assay to test the protein expressed in the transfcted cells. Your ultimate goal is to check if siRNA or shRNA perform their jobs biologically in transfected cells

Hi Gianluca,

I believe all these answers helped you a lot to decide. I would also first try transient transfection and then continue for the stable expression of shRNA. You will need viral vectors such as lentiviral or AAV for stable cell lines. Just to add my two pennies, all these transfection depends on the cell type you are using. Some cells are very happy to be transfected; however some are very stubborn for this procedure. You may need to try several methods of gene transfer. The key for a better shot is expanded literature review. You may find someone already did it and kindly ask for a stable cell line.

Good luck with your study.

Hi!

For cell line, stable transfection would be better. siRNA is a better choice for primary culture when cells could not be cultured for longer time.

Good luck.

If your target gene is vital for cell survival, then a stable transfection will likely not work as those cells where knock down is achieved will die (or stop proliferating), then the transient transfection is best but then you need to optimise your transfection procedure. If your target gene is not essential you can always go for the stable transfection, note that sometimes once you do that, cells have been selected to compensate for the lack of the target gene by tuning the expression of some other gene/pathway so the interpretation of the results might be compromised.

Hi Gianluca,

I think that the stable transfection with shRNA may be a good point to start with. Depending on your aims and also on the journal you want to target you can go to more sophisticated methods such as inducible constructs, gain of function experiments or in vivo with KO or conditional KO if it is available. Good luck!

Hi, I think if you start with siRNAs transient transfection, might try co-transfect target gene reporter vector (like gene fused with GFP). At this point, you can co-transfect any type of cells, 293T, Hela et al. After you verified transfected siRNAs on GFP expression, you could clone the same siRNA sequence to the stable vector.

good luck with your work.

Your experiment sounds like shRNA would be preferred for two reasons. Primarily since your cells are hard to transfect you would benefit from a vector based system that includes a selection marker (e.g. puromycin). If you are only seeing 20% transfection efficiency a selection marker will help you remove the other 80% of cells interfering with your read out. For example, even validating knockdown would be difficult because you could have 100% knockdown in 20% of the cells and 0% knockdown in 80% of the cells. When you measure knockdown in a population where there are so many cells without the plasmid your knockdown will appear to have failed. After selection you will only have cells that have taken up the plasmid and it will give you a much cleaner readout.

Also, whether or not you need a stable cell line should be determined by your phenotype or experimental design. If you can see a phenotypic change in a short period (1wk) then transient transfection could work. If you need to take the experiment out to later time points then you want to consider a stable cell line. So, you may not need the upfront work of creating a stable cell line.

Many shRNA plasmids are adapted so that they not only can be transfected as a plasmid, but they can also be packaged into virus to transduce cells. So, this gives you options for short term transient transfection with plasmid or stable cell lines for longer term experiments from plasmid or more efficiently from viral transduction.

As a disclaimer, I work for this company, but I highly recommend you look at transOMIC technologies shRNA system. The targeting sequences are chosen based on a unique new design algorithm. Without going too far into it, every construct is expected to knockdown your gene by >70% even if it is at single copy in the cell (also a benefit for hard to transfect cells). So, they are very potent and you don't have to purchase extra shRNA to test in hopes that a few will work.

I would agree with Giuseppe. Lipid-based siRNA transfection is usually very efficient and what is also important - easily scalabe ( you can "dial" you knock down simply using siRNA concentration). Knockdown using shRNA is largely much less predictable due to genomic background variations between clones in case of stable cell lines. It is also less advisable due to lower transfection efficiency with plasmids (in case you are not using lenti).

I advise you to star with transient transfections, it's way much easier and faster. If you would like to knock-down a gene with siRNAs, then used LIPOFECTAMINEIMAX, it worked perfectlyfor me with two different primary cells with 90% silencing of the gene. Good luck!

Thanks for all your answers. I tried lipofectamine2000 and oligofectamine. Both of them work.

Thank you very much to all of you now I have clearer ideas. I think I'm ready for approaching stable transfection. I ordered shRNA from SABiosciences and I will transfect all my cell lines with lipofectamine 2000. I was able to increase transfection efficiency to 50% changing liposomes concentrations, which should be ok.

Thanks again

Gianluca

An shRNA controlled by an inducible promoter would be ideal for you. This allows you to switch back and forth and allows the analysis of the same cell population. It requires lot of work up front. However, in the long run it would be worthwhile.

I would go for stable transfection and that may be by CRISPER/Cas 9 technique if I want to pursue the role of the specific gene in vivo animal model. Further, this may get me info for survival, recurrence, and more info of gene if it has a role in prognosis,

It depends on your objectives and long term aims. For getting quick answers, siRNA/shRNA may work for all your in vitro experiments.

Gene silencing can be achieved through either stable or transient transfection, and the choice between these two methods depends on the goals of your experiment or study.

In the context of gene silencing specifically:

• Transient approaches are often used when the goal is to study the short-term effects of silencing a gene. This can be achieved using methods such as siRNA or short hairpin RNA (shRNA) transfection.
• Stable approaches are used when long-term silencing is needed. This usually involves integrating shRNA or microRNA constructs into the genome, often using viral vectors, and selecting for cells that have stably incorporated the construct.

Ultimately, the choice between stable and transient transfection for gene silencing depends on the specific requirements of your research and the duration over which you wish to observe the effects of gene silencing.

l With this protocol list, we might find more ways to solve this problem.