I am doing a ddPCR GEX project using HPSE gene. I have done many runs with different parameters. ( i have done the temperature gradient to optimise temperature), i have done several runs with various cDNA concentrations, got a new HPSE probe in to make sure that i didn't have a bad probe/primer.
The samples i am using in the pictures attached Wells A01- D01 and G01 should have high HPSE expression and E01, F01 lower expression. But nothing is showing any expression. The reference gene is working so i don't think it is a sample issue(i could be wrong).
Can anyone provide any advice on what i am doing wrong or what the issue could be? The probe/primers are purchased off the shelf from biorad. Should i design my own and try, or do you think the issue will be the same.
Thank you.
There is no problem with your sample the way you explained it. However, you need to work on your probe and primers. Please seethe links below could help you to sort out your problem.
https://www.nature.com/articles/2404821
https://www.biorxiv.org/content/10.1101/2020.03.26.008730v1.full.pdf
All the best !!
Thank you Anju Kaushal after several emails and meetings with biorad they have concluded that is the primer/probes they sent me. Now I will be designing my own and hopefully have a better result.
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