I have a question.
Assuming I have the following dilution series with known control.copies/uL 200000 20000 2000 200 20 2
My question is, should I add this to my RT-qPCR reaction 4X concentrated to reach the same copies/ul to the advertised or it does not matter?
I ask because I was told to use 5 uL from each dilution and my RT-qPCR final volume is 20 uL.
Therefore, if I add 5 uL of the first point, this is 5 x 200 000/copies = 100,000,000 copies in final 20 uL or new concentration of 50,000 copies/uL in the reaction.
If I did this way, should I report my results according to 50,000 copies/uL or 200,000 copies/uL?
The alternative is to add 5uL of the dilution series 4X as concentrated, so when I add it to my reaction it dilutes and stays at my initial target number.
I read a lot of protocols and no one mentions this detail.
Thank you very much.