I have run multiple qPCR on my water and sediment samples. Every time I run, I get different Ct values, which means the number of copies also changes (by a factor of 10). What might have happened? I kept the reaction settings the same.
Hi,
Firstly, are you running a standard curve alongside your samples? Check the individual Cts and the efficiency of the qPCR from the slope. Shifts in Ct can be due to suboptimal run conditions or a problem with your mastermix (degraded enzyme etc.). If you notice that your standard curve and your sample wells Ct values have similar gaps between the before/after run (e.g. always 4-5 Cts different to each other), this signifies that the PCR assay itself has a problem and not your sample.
If the issue is only within sample wells, include PCR inhibitor controls if possible or dilute the DNA template as necessary (e.g. 1:10) and run again.
I think the varying Ct values have to do with changes in DNA integrity or quality. Are you using the same DNA sample every time? that is, is your DNA exposed to repeated freeze/thaw cycles? you should aliquot DNA to avoid that. Regarding the standard curve (assuming you are running an assay with an external standard curve) you should use a fresh preparation each time, which you can prepare ahead and store at -20°C but should be used only once.
I am not even sure about my DNA quality. When I check the concentration with Nanodrop, the concentration was below 5 ng/uL (~2 ng/uL). I just have a look back to my analysis. It is true that when I use different aliquots of the same DNA (prepared on same day), the Ct values and # of copies seem to closer within 2 runs.
@ Nadine Gund: What type of error are you thinking of? Replication or I pipette wrong amount of DNA into the tube?
@Amin: My sample concentration is very low already so I can't dilute them down more. But if I do, how it would make the different?
It is important that you check DNA quality by agarose gel (1%) electrophoresis as well as quantity by Nanodrop (2 ng/ul is acceptable for qPCR). You may see in the gel whether DNA is badly sheared or not.
http://www.sabiosciences.com/newsletter/TechNote_PCRArrayPipettingError.pdf
http://blog.biosearchtech.com/TheBiosearchTechBlog/bid/43104/Pipetting-for-qPCR
@Maria: I check DNA on the gel and it didn't have any clear band on the gel, most of them are light smears
@Nadine Gund: Thank you
Hi,
Firstly, are you running a standard curve alongside your samples? Check the individual Cts and the efficiency of the qPCR from the slope. Shifts in Ct can be due to suboptimal run conditions or a problem with your mastermix (degraded enzyme etc.). If you notice that your standard curve and your sample wells Ct values have similar gaps between the before/after run (e.g. always 4-5 Cts different to each other), this signifies that the PCR assay itself has a problem and not your sample.
If the issue is only within sample wells, include PCR inhibitor controls if possible or dilute the DNA template as necessary (e.g. 1:10) and run again.
In addition to previous suggestions i can add :
The sample concentration is low ! sampling errors may happen but i think the internal control may help you to eliminate random and systemic errors ..
I think you could tye to increas the sample DNA, for example 2ul, to reduce the pipetting error; and useing a appropriate internal control. If the Ct value difference between internal control and target gene is similar in differnt repeats, the result is believable.
@Gholamreza and Haihai: You mentioned about internal controls, I learned a little bit about it in my Chemistry class but how can you apply it in running qPCR?
You could find some internal genes in the papers online. Then synthze the gene-specific primers and decte in you expremental tissues by qRT-PCR. Excepte the expression constancy, chosening an internal control with the close expression level to that of target gene was the best choose.
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