I have been using the kit above for some time now, which performs cDNA conversion and normal qPCR in the same tube. I am noticing that despite using recently calibrated pipettes and preparing my samples as mastermixes I am getting poor agreement between replicates (> 0.5 Ct difference in most cases). I am now wondering if doing a traditional two step reaction might improve the situation and was wondering if anyone has any advice or experience to comment.
Dear Lindsey, have you tried another PCR instrument? Or are you sure it has maximally possibly even heating of the block?
I have not tried another instrument because we have calibrated with a calibration plate and the data were ok. This does suggest an error in sample preparation, but I don't know how to improve on this since I am using mastermixes for everything. Our pipettes have also been calibrated they recently. Thanks for your help.
What do you mean when you say "replicates"? I assume you mean you're doing three technical replicates, that is, the exact same template pipetted into three different tubes and ran at the same time. Is this the case?
When you say you're using a master mix, do you mean you add your template to a master mix and pipette that into three different wells? Or are you distributing your master mix and then pipetting your template individually three different times? If it's the second method, what volume of template are you adding to each reaction?
Hi Andrew,
By replicates, yes, I mean technical replicates (x5). I prepare a mastermix of everything (including template) and pipette this into the five replicates (in tubes not a plate). This is why I cannot understand why such great differences should be observed between these identical mixtures.
Thanks,
Lindsey
If you can't pipette the exact same solution into different wells and get similar results, that is indeed a problem. Maybe you're having machine problems. My only other idea is to use a reference dye like ROX in addition to the normal kit dye (SYBR?). There should be many protocols for this if you search. Add a second channel for ROX in the qPCR cycling procedure (you're detecting two dyes now, not one). This is a bit of an older strategy, but it basically adds an internal standard for fluorescence detection to the master mix. It might help.
Hi Andrew,
If I understand you correctly, in that you mean to use ROX in the reaction mixture to account for fluorescence variations due to volume fluctuations or other factors, I use the qiagen one-step SYBR green kit which already has ROX in and so does this already.
I reached the same conclusion as you regarding the machine as the situation does not make sense - but the machine gave consistent results for the manufacturers calibration plate. I am utterly confused.
Thanks again, Lindsey
Ok, this might be silly, but how does the ROX look from well to well? I mean, is it constant in every sample or do you see variation between runs with the ROX?
Actually, I have noticed that in the occasional well, within the same run, the ROX is going wild. Some of my more crazy replicate tubes have definitely had unusual ROX data. I haven't paid a lot of attention to this, so I may have only noticed the more extreme examples, but this has occurred at least a couple of times (I am not in the lab currently so cannot look at my data right now).
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