AlexanderA

Hello there,

I am facing a problem with a new Primer set I designed and I hope that someone can help me with this.

I designed primers for SYBR Green qPCR and ran a PCR with temperature gradient from 57-62°C.
The Primers are blasted and are specific for my sequence. One splice variant is also amplified which has the same amplicon size.
The no template control and no rt control did not show any amplification.
I see a broad peak in the melt curve of my product at many temperatures. At some temperatures the meltcurve looks good to me. I ran the products on a 2% Agarose Gel.
Multiple Bands larger than my specific product (120bp) can be seen on the Gel.

Is there anybody who can help me out with this?

Best regards Alex

Hello there,

I am facing a problem regarding qPCR quantitation, and I hope, that someone in this forum might help me.
Currently I am working in a research project in which we perform a gene expression study of human monocytes. There is one group of critically ill patients, and another group which serves as a control group. However I want to perform both quantitation via deltadelta CT method in case PCR efficiencies are comparablem, but also quantitation via relative standard curves.

Now my question is: Which material is suitable for relative standard curve quantitation. I am not interested in absolute quantitation because I am comparing two groups together, but I am interested in how much mRNA content is in the cells independently regarding pcr efficiency.

Would it be appropriate to use THP-1 cells for reference rna?
Best regards

Alexander