Did you specify the concentration of the standards? The other option would be to increase the threshold, on the first image it looks like all your curves start *above* the threshold, maybe the software interprets it as negative

Dear Soufiane Rabbaa,

Verify the quality of your template: Ensure that your template DNA or cDNA is of good quality and not degraded. Use high-quality DNA extraction or cDNA synthesis methods to generate reliable templates for qPCR.

Dilute the template: If the initial concentration of your template is too high, it may inhibit the qPCR reaction. Dilute your template to an appropriate concentration to avoid inhibitory effects.

Check reagents and reaction setup: Confirm that all reagents, including the qPCR master mix, are properly prepared and added in the correct amounts. Verify that the reaction setup, including pipetting and mixing, is accurate and consistent.

Assess instrument settings: Double-check the instrument settings for the Ariamx qPCR system. Ensure that the correct fluorescent channels are selected for your fluorophores, and the instrument is programmed to collect the appropriate data during the amplification process.

Check primer and probe design: Ensure that your primers and probe are designed correctly and specific to your target gene. Validate the primer/probe sequences using tools like Primer-BLAST or Primer3. Confirm that the melting temperatures (Tm) of the primers and probe are within the appropriate range for your qPCR conditions.

And some time it is really important to check the calibration to avoid the errors.

I hope this will help you to evaluate your problem, all the best for your future results.

You need to tell the program the concentration (relative or absolute) for the standards for it to generate a graph.

Also, your amplification is really weak for all of your samples. Just by eye, I'm not seeing good separation of the standards by Ct value. Take a step back and make sure that your template is the correct concentration.

Most folks make "single experiment aliquots" of their standards, freeze them, them them just ONCE and then toss any leftover. Freezing & thawing will degrade your standards and will make amplification less efficient.

Good luck!

Dear Soufiane Rabbaa

Add a non template control (NTC) in one PCR vial and test it.

A non template control is leaving the sample without cDNA. Usually NTC is used to check whether your cDNA is contaminated or to check primer-dimer formation. Here you can use the NTC to rule out which parameter (template quality, primer,reagents,dye) is to be rectified and troubleshooted.

All the best