Undiluted cDNA likely contains PCR inhibitors (carryover salts, ethanol, RT reagents, too much template) that block polymerase activity. A 1:10 dilution lowers inhibitor concentration to a tolerable level while keeping enough target, so qPCR works consistently.

Consider it a win that you have to dilute out your samples. Otherwise you will quickly use up all of it.

Don't bother tracking down the "why" you need to dilute. It's a common phenomenon, as noted by Athmaja V Christophy

Whoever is teaching you how to set up qPCR should have told you about this already.

Just proceed with your experiment. Good luck!

Hi, besides what has been said, also too much DNA has inhibitory effect on DNA polymerases.

Everyone else has given you excellent replies, so I will just add the following:

1) ALWAYS dilute cDNA for qPCR. For all the reasons specified by others here. Concentrated cDNA has a ridiculous number of targets: I use mine at 1:20 dilution and I work on some low abundance genes. I can detect them just fine.

Also remember that qPCR is an exponential process, so cDNA that is 10x as concentrated will only lower your cycle threshold by ~3.3 cycles. There are very few scenarios where 'concentrated cDNA' is a thing you need.

2) RNA quality: I assume you spec your RNA, but do you have good 260:230 ratios? This one is critical for ensuring successful reverse transcription, since high A230 is indicative of guanidium carryover, and guanidium salts will kill your reverse transcriptase quickly (RTs are very delicate snowflake enzymes). Note that 260:280 is far less important.

Aim for 260:230 > 2.0, though depending on your cDNA kit, you might get away with >1.7.

3) Actual raw numbers: what are they telling you? If you have consistent Cq values of ~20 for your reference, that's telling you it's an abundant target which appears to be stably expressed. This is a good thing.

If you have variable Cq values for your GOIs, your interpretation will differ based on what those Cq values actually ARE, and whether they vary between replicates or between samples.

qPCR cannot measure less than 1 target, so there IS a lower limit of detection (usually Cq ~35).

There is also a lower limit of quantification, which requires greater target abundance (Cq ~28-30 or so): you can have targets that are detectable but not readily quantifiable, because with very small numbers of target molecules, you can easily get uneven partitioning between replicate wells. With Cq values greater than 28-30, high variability between replicates is expected. You can address this by using MORE replicates (the average Cq of six replicates is much closer to the real value than the average of only three), but you could also consider whether you have meaningful amounts of target.

At Cq 30-35, your conclusions are "I have almost no target, my GOI is probably not meaningfully expressed in this sample"

If you have robust expression (Cq 20-26 or so) but see variation between samples, this might actually be telling you that expression of your GOI changes between samples.

If you have robust expression but see variation between replicates, conversely, then you probably have bad primers, or need to calibrate your pipettes.