Hello, Thanks for the protocol. And I have some questions. Firstly,...

Firstly, I am curious about the exact transfection efficiency about this method since you use the EB to be transfected directly if I am right.

Yes, you are right. We optimized the standard transfection protocol using EB. To quantify transfection efficiency we performed qPCR on dissociated EB (please, see point 12 of our protocol) and using both a positive control (100% of miRNA expression) and a negative control (P19 naïve cells). RNA was extracted from the isolated cells and RT-PCR and qPCR were performed for miRNA1, miRNA133 and miRNA499. The transfection efficiency ranged 88 to 100%.



Secondly, is the high transfection efficiency just limited to this cell line or the precursor miRNA. I am wondering how is it for the other ESC lines and the general plasmid?

P19 cells are not easy to transfect with liposomes and plasmid. However, the low molecular weight of miRNA precursors (60-80 nucleotides) probably facilitates the transfection efficiency. We initially tried to transfect these cell line with a 10 Kb plasmid but the transfection efficiency was very low. For these reason, we switched to lentivirus to transform P19 cells obtaining high efficiency (please, see Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).
We did not test ESC lines in our studies so we do not have an answer for this specific question.



Thirdly, I am wondering what is the difference between the cardiomyocyte differentiated from this embryonal carcinoma cell line and the normal ESC line? If there is no difference, this cell line is really convenient for cardiac differentiation. And how much percentage of the cardiomyocyte in the EBs differentiaed from this cell line?

As aforementioned, we did not perform the transfection protocol on ESC. Anyway, ESC and P19 behave very similarly and we supposed that the cardiac differentiation efficiency could be similar between these two cell types. Please, refer again to our manuscript (Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).