Can you provide some Ct curves and the Ct values? As well the melt curve graph.

From my personal experience, many published primers are not optimal. You may try designing your own primer pairs. I always use Primer3Plus with the qPCR settings activated and almost always get good primers.

Another issue is how to make a good standard curve. It is not difficult but there are some tips that can increase the quality. First, avoid using small volumes (use at least 50-100 µL for each dilution) since the error of smallest pippetes is much higher. Second, vortex 5-10 seconds before taking the volume for the next serial dilution.

I hope this works for you. Good luck!

I agree with Daniel Martín published primers are not always great. I would try designing your own and ordering a few pairs to test. Another thing you can try is varying the annealing temperature some or adding slightly more primer concentration. However, it is usually easier to mitigate when efficiency is too high rather than too low.

I also always add 2ul of my cDNA to my wells so that I can see it hit the line on the pipet tip for better assurance of accuracy.

Thank you for your suggestions Daniel Martín Julie Ann Dougherty . I am just curious however, in this niche of study, the primer pairs are widely used in many studies and journals, yet it seems like most managed to achieve great pcr efficiencies.

I was wondering if there are other perspectives to improve aside from the primers sequence? Something I could try to improve before giving up and order new set of primers?

Modification to bases like addition of LNA to specific bases could prove useful.

Can Justin Kiessling i've never heard/tried that before. perhaps you could provide me a general description on how to do it?

Adding LNAs are a bit tricky, there are some general rules to follow. Addition of LNA bases will modify the annealing temperature so you will have to keep in mind your PCR protocol can be altered and its impacts. Primer sequence is a major determinant of PCR efficiency, but remember there are also other variables such as carry over contaminants from isolation, PCR buffer formulation and annealing temperature. It seems like soil is your sample type which contains inhibitors, if your isolation kit doesn't deal with this then perhaps your downstream reaction can be inhibited (https://npdn.org/newsletter/2020/12/article/3#:~:text=Soil%20contains%20humic%20acid%2C%20which,or%20reduce%20PCR%20inhibitors%20considerably.). Before going down the LNA route, I would recommend looking at these preanalytical variables and even re-consider primer design like the above suggestions. I quiet enjoy primer design for SYBR green assays. It is definitely a skill worth having.

Jasmine Keller Efficiency can also depend on the qPCR master mix and the machine you are running them on. the salts and concentrations of dNTPS can vary and the machine ramping/cycling can be different.

Also your RNA quality will affect the reaction so if your 260/230 is low or if you run a BioAnalyzer and the RIN is low then that will affect efficiency also.

it's a lot of small things but try to focus on controlling what you can to get it to work in your hands.

Sometimes you can't increase the efficiency, you just have to design new primers.

That being said, the extremely high and extremely low ends of your standard curve can throw off your curve. It's OK to not use all of the standards as long as the range of the standards is inclusive of all of your unknowns.

Good luck!