In one text (wallace and Rieske 2023) I found this "Quantitative PCR was then performed on a 5-fold dilution of cDNA (starting at a 1:25 dilution) "while on the other paper (Sellamuthu et al., 2022) i found this not explicitly mentioned the ratio or the dilution factor , how to find this or decide which is better for my sample"cDNA samples were diluted ten times before RT-qPCR reaction."
To determine the optimal dilution factor for qPCR, start by assessing the initial concentration of your DNA or RNA using a NanoDrop, Qubit, or spectrophotometer. Ideal starting concentrations typically range from 1–100 ng/µL for cDNA, 1–10 ng/µL for genomic DNA, and 10–100 ng/µL for RNA in RT-qPCR. Highly concentrated samples may contain PCR inhibitors such as heme, salts, or proteins, which can affect amplification efficiency. To check for inhibition, prepare a serial dilution series, such as 1:10, 1:20, 1:50, or 1:100, and observe how the Ct values shift. Creating a standard curve from a dilution series also helps determine assay efficiency, which should ideally be between 90–110% with an R² value greater than 0.98. If the efficiency is too high or low, the template concentration may need adjustment. The level of gene expression also plays a role in dilution selection—highly expressed genes require greater dilution (e.g., 1:100 or 1:1000), while low-expression genes need minimal dilution (e.g., 1:2 or 1:5). A good practice is to aim for Ct values between 15 and 35; if Ct is below 15, the sample is too concentrated and should be diluted further, while Ct values above 35 indicate excessive dilution. Running a small pilot qPCR experiment on different dilutions can help determine the best concentration, ensuring minimal variation between replicates (standard deviation ≤ 0.5 Ct) and achieving efficiency close to 100%. Selecting the appropriate dilution factor is crucial for accurate, reproducible, and reliable qPCR results
Honestly, just "always dilute your cDNA" is a suitable response. cDNA synthesis involves all sorts of things that are deleterious/inhibitory to downstream qPCR.
I would recommend 1/5 at the very least, using 1ul of that diluted cDNA per reaction. I would not go higher than 1/20, though, unless your target genes are incredibly abundant.
If it helps, I use 1/20 dilution, and then use 2ul of diluted cDNA in a 10ul reaction, so essentially 0.1ul of my original cDNA.
I used to use 1/10 and 1ul, but I've found that 2ul is a much easier volume to reliably pipette.
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