The exact ng will depend on the expression of your genes in your exact samples. You can check publications with similar experiments to get an idea of the range. But the only way to know for sure is to try your samples. The kit has suggestions for how much to start with.

You'll have to know something about the lower detection limit from your standard curve. You can increase the amount of starting ng for your genes-of-interest and account for the change in starting materials for your calculations.

Good luck!

It's a bit of trial and error. BMPs can be tricky sometimes. Usually, we do the RT with "roughly" the same amount of RNA per sample. Then we dilute the samples in the same way to reduce variability.

My go-to is 200-300 ng of RNA in the RT, then diluting in whichever way it's convenient to have around "5 ng" of the original RNA per well.

If your CTs are too low after the first trial you can always increase the cDNA template or the RNA you use in the first step.

Dear Panagiotis N. Stoikos

You can use 4 to 10 ng. For genes that have high expression levels such GAPDH, even less of that amount is fine.

You can use 4 to 10 ng. For genes that have high expression levels such GAPDH, even less of that amount is fine. t60/230) must be 2-2,2 and even upper than 1.7 is enough. If you have a low value the best solution is to dilute your sample, in my experience at least 1:100. If your samples have a good quality then you have to think about other inhibitors such as the massive amount of RT ....etc. If no way was useful it would better to re-synthesis cDNA.

Best...