Hello Denti Rizki Kinanti

On the one hand, answering your questions:

1. The quality of the plasmid extraction is correct. 1.99 is perfect. In my opinion, this does not affect the essay. But you have to consider very well is the concentration of the plasmid, to make correct dilutions.

-If the insert, target of the PCR, that you have inserted into the plasmid, does not have an upstream promoter that makes it express itself. And if what you are going to do is a PCR (not an RT-PCR), I DO NOT think it is necessary to do a treatment with RNAses.

2. If what you are going to quantify is DNA (plasmid + target), I DO NOT SEE NECESSARY that you include RNAse inhibitors and RT.

One piece of advice, if you are going to develop an absolute quantification method, whose real target is RNA, my recommendation is that you also design the standard curve with RNA. For this, you have to express your insert from the plasmid using an RNA polymerase. If you have doubts about how to do it, you can consult a publication of mine where I did something similar.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197237

Another tip is to make sure that you are having amplification of your target, for this you can run the one amplified by qPCR in a gel.

If my answers have helped you, don't forget to click "recommend"

Regards ;)

I think you would need to find optimized primers that do not have any dimers.

Hello Denti Rizki Kinanti

On the one hand, answering your questions:

1. The quality of the plasmid extraction is correct. 1.99 is perfect. In my opinion, this does not affect the essay. But you have to consider very well is the concentration of the plasmid, to make correct dilutions.

-If the insert, target of the PCR, that you have inserted into the plasmid, does not have an upstream promoter that makes it express itself. And if what you are going to do is a PCR (not an RT-PCR), I DO NOT think it is necessary to do a treatment with RNAses.

2. If what you are going to quantify is DNA (plasmid + target), I DO NOT SEE NECESSARY that you include RNAse inhibitors and RT.

One piece of advice, if you are going to develop an absolute quantification method, whose real target is RNA, my recommendation is that you also design the standard curve with RNA. For this, you have to express your insert from the plasmid using an RNA polymerase. If you have doubts about how to do it, you can consult a publication of mine where I did something similar.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197237

Another tip is to make sure that you are having amplification of your target, for this you can run the one amplified by qPCR in a gel.

If my answers have helped you, don't forget to click "recommend"

Regards ;)

qPCR assays are challenging to design. Most of the primers that you test will not have high enough efficiency to work. I'd suggest designing several pairs and start by testing their annealing temperature on a standard PCR with a gradient. If the primers don't give a strong band at the annealing temp used in qPCR, then they simply won't work well.

When setting up a standard curve, there is a limited range of concentrations that are useful. Too many copies are generally worse than very few. I've been able to get a linear slope & excellent efficiency with the low end of my standards at 10 copies/microliter.

But the most likely explanation is that your primers just are efficient enough. Try designing some more.

Good luck!