before we doing the relative quantitative real-time PCR, we should generate a standard curve. when we are generating the standard curve what should we pay attention to to make your curve more precise?
- Do serial dilution of standard, starting from high to low conc.
- Increase the pipette volume by more dilution of standard sample. Say if you have 10 ng/microliter and you have to put 1 microliter to get 10 ng but if you dilute the sample to 2.5 ng/microliter you have to put more volume i.e. 4 microliter.
- You need to add standard every time you run qPCR
Good luck.
the repeability for each dilution gradient is not very good. sometimes, the Ct value is even bigger for the hihger concentration one than the lower concentration. is there something wrong with the dilution? what should I do to improve the result of the standard curve?
According to my experience making individual dilution increases the pipetting error when the standard concentration are low but it is highly reduced when you go for serial dilution
say 1000 ng/microliter as starting concentration, you can make
- 10-1 diln by taking 10 microliter of 1000 ng/microliter and add to 90 microliter nuclease free water.
- 10-2 diln by taking 10 microliter from 10-1 diln and add to 90 microliter nuclease free water.
And so on until your get your desire range.
I have done the above protocol, got good and consistent result.
See the amplification curve attach.
Yes 3 technical replicate for each concentration range is enough. If you are confident about your pipetting skill, you can even use duplicate.
I suggest to try wide range of concentration to figure out which range is giving near 100% efficiency and use that range.
Hi Liang
I would like to add one thing. Since you mentioned your higher concentration show higher Ct I believe you are having a problem with an inhibitor. Can you descard that concentration? If you remove it does your linearity improeve? Is your test sample in the middle of the curve?
waht do you mean? I do not understand your meaning clearly? can you explain further?
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