Hi Bernardo,

It depends on your cDNA quality, target gene and PCR efficiency which you can find out it by serial dilution test. We performed some experiments with 1 ng of cDNA per reaction.

I recommend you to prepare serial dilution base on cDNA concentration to find the optimal concentration to have higher PCR efficiency. Since you want to use ΔCt method, PCR efficiency is so important.

As much as required to have a minimum of about 100 target molecules per reaction (below this amount the Poisson distribution of molecules makes a precise quantification difficult).

Hi Bernardo,

It depends on your cDNA quality, target gene and PCR efficiency which you can find out it by serial dilution test. We performed some experiments with 1 ng of cDNA per reaction.

I recommend you to prepare serial dilution base on cDNA concentration to find the optimal concentration to have higher PCR efficiency. Since you want to use ΔCt method, PCR efficiency is so important.

More is not necessarily better. With PCR, large amounts of nucleic acid can sometimes gunk up the reaction, so yes -- as the last person suggested. Try a few concentrations until you are in the middle of your standard. It comes a little with experience with your system. I tend to think that the lower quantitative limit is more like a 1000 copies, though 100 is frequently used as suggested by JW.

For a 20 uL RT-qPCR using the ΔCt-method I have used up to 2ug of RNA, but it really depends on how well your gene of interest is expressed. As Somayeh Jamali suggested, it may be worthwhile to perform a serial dillution to test which amount is best.

8ng cDNA is usually sufficient for highly expressed genes, like housekeeping genes (we use GAPDH and 18s). A serial dilution test would put you in the correct range.

Hi, Bernardo

How much cDNA was used in the PCR depend on PCR reaction system, the expression of your genes and the brands of the reagent. your should try a serial dilution test to find the correct quantity.

Another thing you should pay attention. When you conduct the reverse transcription, the same quantity of total RNA should be used in all reaction. In our study, 400ng total RNA was used in 10ul reaction system using takara RT reagent kit.

Hi Bernardo

To all what was said before, I would add that, it is good to work with dillutionsof cDNA which give the Ct values between 25 and 30, and obviously keep the same quantity for testing all the genes.

Hi all, 

If I have a concentration of 15ug/mL, so I should do serial dilution, with 15ug/mL as the biggest concentration, right? ANd other concentration would be, 7.5ug/ml, 3.75ug/ml, 1.875ug/ml and 0.9375ug/ml? Thank you in advance.