Hey everybody,
I did a qPCR and gained results, however in some of my samples, one of my triplicates has a much higher Ct value than the others. This is quite frequent but only just one sample is showing this pattern, I used the same cDNA the same SYBR green same primers for each reaction, I wondered why this could be, would bubbles have an effect or is it solely on my technique. Additionally the program generates the standard curve automatically, so I must generate the graphs manually any tips on how to do that? Appreciate the help.
Regards;
Ege
Hey Ege, multiple things come to mind. Here are some ideas:
Are the high Ct values in the same row or column? In that case I would suspect your SYBR optics to be broken. We've had a similar issue on our Eppendorf machine a while ago. Or, in case you are using a multi-channel pipette, it is possible that it is not calibrated right.
Bubbles could be at fault too. It is always a good idea to spin down your plate in a centrifuge after you have sealed it and before you put it onto the qPCR machine (I just do 30sec @ 3000 rpm). That usually gets rid of bubbles.
Are the high Ct values on the outside of your plate? Then your machine could also not be calibrated correctly. As the outer edges of the heating block tend to be colder than its inside, there will always be some variation (usually higher Ct values) there. However, for most machines that should not make a big difference (
Hey Ege, multiple things come to mind. Here are some ideas:
Are the high Ct values in the same row or column? In that case I would suspect your SYBR optics to be broken. We've had a similar issue on our Eppendorf machine a while ago. Or, in case you are using a multi-channel pipette, it is possible that it is not calibrated right.
Bubbles could be at fault too. It is always a good idea to spin down your plate in a centrifuge after you have sealed it and before you put it onto the qPCR machine (I just do 30sec @ 3000 rpm). That usually gets rid of bubbles.
Are the high Ct values on the outside of your plate? Then your machine could also not be calibrated correctly. As the outer edges of the heating block tend to be colder than its inside, there will always be some variation (usually higher Ct values) there. However, for most machines that should not make a big difference (
Hi Ege,
Since you are having this problem with only one sample, make sure the DNA is well diluted in the water or TE. Sometimes we had problems with samples that had so much DNA it did not dilute well. This led to the problem that when pipetting, you could carry high amounts of undiluted DNA to you final reaction.
As Felix suggests, I found that spin down your plates in a centrifuge is essential not only to get rid of the bubbles, but also to be sure all reagents are mixed properly.
About graphics I make them manually since they are often more appealing than the defaults you get with RT-PCR machine software. I use Python with Pandas since I don't feel comfortable with R, but any of them will help you.
If you are using a Roche Lightcycler 480, I wrote modules and scripts to process its outputs and get better figures.
Bests!
Two quick points:
1.) Be sure to prepare 1 tube containing each of your triplicate replicates, and pipette out of that tube into the plate. (Do not set up triplicate replicates directly in the plate).
2.) Plotting log of dilution factor vs Cq in Excel will allow you to use the machine Cq values to manually generate your standard curves. Be sure to use a valid stable threshold on a target by target basis when harvesting your Cq values.
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