Muhammad Hafeez: To my knowledge, I have seen people facing the same problems sometimes. In most cases, it might be due to DNA contamination. You might have picked DNA also during aqueous phase separation while separating RNA. Another reason can be primer dimer also. From your question, I guess you are getting some values lower than your housekeeping gene Cq value, which mostly means DNA contamination. If it is a primer dimer, you check your qRT-PCR data's melting curve. If you see two peeks in, it means primer dimer or DNA contamination. In that case, I suggest you do DNAse treatment of your RNA samples, from which you can convert cDNA again and run qRT-PCR. I hope this will help you to resolve the issue. Muhammad Hafeez

Here are a few potential reasons for this issue: Contamination, primer-dimer formation, Non-Specific Amplification, Carryover Contamination, etc.

To troubleshoot, run a no-template control (NTC) alongside your negative control to check for contamination. Optimize your primer design and PCR conditions to minimize primer-dimer formation and non-specific amplification.

Which negative control(s) are giving you bands? If it's the no-RT, then you probably have DNA carryover/incomplete DNase treatment. Check the sizes of the products on an agarose gel.

If you have amplification in your "no template, just water", then you have contamination (or, small chance it's just primer-dimers). Again, run out your samples on an agarose gel to see what size product(s) you amplified.

If it's contamination, you need to throw away ALL of your reagents (and I do mean all of them, even the qPCR mix, the water at your bench, the primers, your TE buffer. All of it). Then, clean your bench. Get brand-new plastic tips/tubes/plates. And clean your micropipettes.

Remember, the goal is to get the assay working, not to chase down exactly why the contamination happened.

Good luck!