I performed a transient transfection of premiR , I have analyzed the efficiency of transfection by flow cytometry after 24 , 48 and 72 hours after transfection through the analysis of cells transfected with scramble fluorescent probe , the efficiency was higher than 80 % in all 3 cases analyzed , a little higher at 48 hours from trasfezion . to verify that actually occurred transfection went to make a real-time PCR on cells transfected with the premiR of my mirna and with those with the scramble , but the real-time data show an increase I of the DCT in cells transfected with the premiR my mirna especially 24 hours after transfection compared to scramble . how can I interpret these results ?
The last sentence is not understandable so giving a suitable answer is not possible because I do not understand what you received by qPCR!
Okay so I read the last sentence for 5 min and now I get an idea what you mean, hopefully.
Often after 24 hours the cells are affected by transfection procedure, you will also see that after the plasmid transfection a part of the population dies. If you harvest then the whole population then these cells with no expression because they are dead cause that the delta Cq value increases > lower expression. In addition the cells containing the plasmid do not transcribe your miRNA 24 hours post tranfection so this cells do not deliver a compensation to the lowering signals derived from dead cells. It strongly depends on the plasmid how long it takes that you see an increase of your insert so it can be a good idea to test sample 48, 72 hours post transfection. The 80% you detected with your colored probe is just saying you that you have a transfection efficacy of 80% and in addition small fluorescent probes tend to diffuse into over time into treated cells causing an "increased" or "higher" % of positve cells compared to cells the medium was changed 4-6 hours (depends on your transfection medium).
Keep in mind that transfecting a small fluorescent probe has other properties than a plasmid with 2-5 kb.
I hope this was helpful.
I didn't understand your question. A higher dCT indicates lower amounts of the target gene. Isn't that what you were expecting by using miRNA?
Yes ture, I have also overseen the fact increased delta CT value then the miRNA should do its job. The last sentence is written a bit confusing.
Hello I'm a bit confuse. When you performed the flow analysis you targeted the scramble fluorescent probe? or the target protein?
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