Hi, so I was just wondering what would be a good Ct value to aim for in real time PCR. My lab typically uses about 150ng of cDNA per well for a 10uL real time qPCR reaction. But in a recent time course treatment experiment, not a lot of RNA was collected and my PI wants to run 13 genes. I didn't have enough cDNA to make triplicates of that many genes and I ended up running a second time course to get more RNA (but that isn't ideal obviously). I read that real time qPCR can use as little as 100 pg of cDNA. The highest Ct that I have gotten with the old procedure for a gene has been around 26. Can I dilute it so that the Ct is around 35 or is that too close to 40 for comfort? I realize that a 2^9 fold dilution is a bit extreme, but this is just for future reference. Thanks.
You can dilute it to get more qPCR measurements, but once you start going past 30 cycles your Ct replicates might not be so tight. I am assuming you have technical triplicates for each biological replicate(s). If you want to see the tightness of Cts, you can find some useless DNA/RNA lying around, do the dilutions to get it to 35 and see with 10-20 replicates of the same sample how the Cts behave around late/mid-late cycles. Imagine the deviation. In my experience, and I've used Eppendorf liquid handling robotics to prepare PCR reactions, once you start going past around 32 cycles even triplicates can start having a tough time keeping a tight Ct range. If you're after small effect sizes, then this isn't the way to go about it. Here's a paper to clarify some of the concepts - https://pubmed.ncbi.nlm.nih.gov/28702366/ (figures 1 & 4 are especially pertinent).
150ng per well is masses.
You are probably getting high Cqs because you're massively overloading the well, and moreover adding too much cDNA synthesis buffer (which is inhibitory to PCR).
Dilute your cDNA. Always dilute your cDNA.
This seems to be one of those things that "everyone knows but never says", so people keep having to discover this the hard way.
For reference, I use ~8ng per well, and I work on some pretty low abundance stuff.
Make your cDNA, dilute it ~1/10 (I dilute 1/20 and then use 2ul per well because I find that easier, but 1/10 and 1ul per well is the same thing), then run your PCRs again.
You may well find that you have much better Cq values. Also, your cDNA will last a lot longer because you have a much larger volume now.
As a ballpark reference, Cq ~35 means you started with probably only one target molecule in that well. You will usually find that Cqs from ~29-35 are pretty variable between wells, because when you're dealing with (and quantifying) countable numbers of molecules, you can easily get uneven partitioning between replicate wells.
If you really want to quantify targets of such low abundance, you can simply include more replicates: the average behaviour of six replicate wells is much more representative than that of three replicate wells.
Hi,
The Ct values varies from one gene/ sample to another. If you are using the Sybr green method, the Ct should be lower than 35 for the samples. In your case, diluting the cDNA can help since the highest Ct you obtained was 26.
Hii Jackson,
The Ct value differs from experiment to experiment. But if you are using SYBER Green then Ct value above 30 is usually not considered. Just remember, the higher the Ct value the lower the concentration of your gene of interest, and vice versa. So, choose the Ct value according to your experiment.
Hope you find it helpful.
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