10 replies to this topic

[Tabaluga]

Hi people,

I'm struggling with a problem in my qPCRs. In every plate I have some wells (and each time different ones) that result in Ct values that do not make sense (e.g. Ct 4 when it should be around 22). The other wells are fine. The curious thing is that I have everything in triplicates and of the three wells it is usually just one that fails ! I have had this problem for a while now and obviously tried to find out whether it is some contaminated stock, but replacing everything did not help. Also, since I do not see a pattern, I cannot pinpoint it to a certain material or procedure step - I have had this phenomenon with different primers, different targets...

But it's the fact that the corresponding technical triplicates are fine which strikes me most, because this means the problem is not explainable by contamination of a certain reagent.

 

Any ideas ? I simply can't find an explanation for this phenomenon. Thanks...

[postdoc]

It appears to me a hardware problem. How long has it been since your machine was last served? Well contamination, sensor failure, sticky things on lid could all be attributed to. If the machine has not been recently served, I'd suggest you order service. It's really worth it. As a temporary solution, you can have quadruplicates for each sample. We do this sometimes even when our machine behaves normally.   

[Tabaluga]

Thanks for your comment. I considered a machine problem too but what doesn't fit is the fact that the "strange" wells are not the same ones through several runs - wouldn't one expect it to be like always well A3 or well H5 or whatever which has the problem then ? Also, other people in the lab don't report this and they use the same machine. The machine seems to be working fine.

About quadruplicates, as you said it might be a temporary solution, but I still need to find where the problem is.

[Dr. Kannan S]

What I can immediately think of is to calibrate your pipettes - if you are adding small volume of cDNA, then even a minute change will give ackward Ct values. you can try using different set of pipettes and check if this problem still exists.

[Tabaluga]

Good point... I did check my pipets, but only once whether the amount is correct and not for consistency. I'll try a different pipet for cDNA pipeting next time. I know it's exponential, but somehow I still find it hard to believe that the resulting Ct difference from small variations will be that high (Ct 4 vs. 22). But I'll certainly keep it in mind for the next run.

[PhalanxBio]

My first hunch was that of postdoc - that some wells are malfunctioning due to hardware, well contamination, etc.  I used to run a lot of qPCRs on a ABI7300 and there were always aberrant Ct values for 2 wells.  It was the same 2 wells every time.  Since you're seeing it in different wells, it definitely points to another problem.  You mentioned that other users haven't noticed a problem - perhaps they haven't looked hard enough.  It would be easy to overlook (and it is unlikely to affect your results) because you're running technical triplicates.  If the 3 replicates are tight, then I don't touch the data.  But if one of the 3 replicates is hugely different, then I get rid of it.  The variability of qPCR and pipetting is exactly why we run triplicate reactions.  I honestly wouldn't spend too much time trying to solve this problem if your other 2 replicates are tight.

[Tabaluga]

Thanks for the comment! I explicitly asked the others about the problem and we are all required to look at the Ct standard deviation anyway in our lab's qpcr analysis sheet, so it couldn't be overlooked. I also wouldn't take these strange values for further analysis and stick with the normal ones, because they are so obviously wrong but still it's a mystery to me (and makes some people think I'm too dumb to pipet correctly....yeah, like I always put a lot more cDNA in random wells and never realise it....the data itself are perfect, but when the plate looks so horrible that makes the whole thing seem less trustworthy and presentable)

[Epigeneticist]

I have seen random well failures when the seal is broken on the clear adhesive sheet. Are you inspecting well volumes post run? Sometimes the volumes are very minimal so it is also crucial to inspect the adhesive sheets for anything that appears abnormal. If this is happening then the best solution is to spend extra time and effort applying pressure to the adhesive sheet, especially around the perimeter until you see no bubbles indicating complete contact between adhesive sheet and plate. This happened to me with the ABI 7500. 

[Mohamed 1984]

I think it is just pipetting error. 

[Poorti]

Hi Tabaluga

 

Could you figure out what was giving you trouble in your qPCR experiments ?

[Tabaluga]

No, unfortunately I didn't. It didn't stop either, so I just had to learn to live with it (i.e. repeat the experiment when it happened).