Hi Hellen,

I assume your AAV carries a gene that is also expressed endogenously by your human cells. Will you obtain cDNAs and permorm the qPCR?

I think your approach is correct (spanning the junction of the FLAG sequence and your gene), just a couple of considerations about it:

1- to ensure more specificity you can design one of your primers to hybridise directly into your FLAG epitope sequence (as your gene will be transcribed with it). As the FLAG sequence is only present in your exogenous transgenic AAV won't cause trouble mixing hibridising with other regions.

2- Your FLAG epitope is only FLAG or FLAGx3? (which is a bit longer) Does your transgene have any extra "linker" regions of nucleotides between the FLAG epitope and your gene sequence? Because if you have these "linker" you can also take advantage and span both the linker nucleotides and the FLAG sequence (some of their nucleotides at least) in your primer design.

Remember that in qPCR the amplicon must not have too much bases (not recommended more than 150 nucleotides) and also the general tips in primer designs (similar melting temperature (Tm) of both primers, avoid self and cross-hybridation between primers due to complementary regions...) that you can find in online tools like ThermoFisher.

If you have other doubts about the primer design do not hesitate to contact :)

Also I would recommend testing more than one viral dose of the AAV (I work with Adenovirus, but not with AAV so surely you are way more skilled with those) and also take into account the cells that are going to express your gene of interest (I faced some differences when I transfected a gene with a FLAG epitope between cell types).

What cells will you use for your experiments?

Best regards! I hope your assays go well

Francesc Estrany

Thank you so much for your answer Francesc Estrany. It is really helpful.

Yes, I plan to get cDNA and do qPCR. One of my primers actually anneal half on the gene and half on the FLAG. the other is on the other side (away from the gene) on the WPRE region.

Regarding FLAG, I have only 1, I hope it will be ok anyway as I could pick it up by immunofluorescence

I will be using HEK293T cells for the experiment.

Thank you very much again for your answer!

Hi Helen Smith

So both the WPRE region and the FLAG epitope are in the 3' region (or the C-terminal of the protein)?

I think that HEK293T won't cause you any trouble, as they are relatively easy to transduce and transfect. And importantly, check that the expression of your transgene is good both in RNA and in protein level! (Once it happened to us that, with an adenoviral vector, our transgene was well transcribed but didn't translate properly.

I hope your experiment goes well!

Best regards

Francesc