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Here we discuss the use and processing of inter-plate calibrators
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4 posts • Page 1 of 1
I have a question regarding the use of the IPC.
It was suggested to me that the use of IPC's can be avoided if you have the opportunity to level the threshold and baseline settings for different runs for each assay. To be more precise: this means that for each assay one would set the threshold and baseline at a suitable value on all relevant plates. This should subsequently be done for all assays. (One setting for all assays is optional, not a necessity).
I'm convinced this method cancels out several variables but I'm not quite sure this could completely rule out the use of an IPC. However, an IPC is also prone to variations and could never completely mimic running the samples in one run. Furthermore introducing an IPC necessitates an extra calculation step which automatically leads to an increase in the standard deviation. Therefore, I feel that setting the threshold and baseline may not be completely identical to using an IPC, but I wonder if the difference between the two methods is significant. Since I currently don't have any data available to test it for myself, I was wondering if you would share your thoughts and experiences on this.
Jeroem, you are absolutely right. Interplate calibration introduces noise and should be used with care. For example, the interplate calibrator shall be purified material amplified with a highly optimized assays. It should also be measured in replicates (at least three). One interplate calibrator per instrument channel is usually sufficient (not one per marker as some companies recommend). In fact, because of the noise introduced by interplate calibration several companies leave out interplate calibrators in their preloaded panels. I don't think that's good idea; maybe they did not know how to design the interplate calibrator properly :-) Highly optimized interplate calibrator can be ordered from TATAA Biocenter, which saves you time, if you don't want to design one yourself.
First of all, many thanks for the answer.
However, I feel like you haven't completely answered my question. In the last paragraph I wrote: "I feel that setting the threshold and baseline may not be completely identical to using an IPC, but I wonder if the difference between the two methods is significant." Am I correct in deducing from the fact that you don't address this specific question, that you don't know of any references on this subject and that you also haven't tested it for yourself?
My apologies for formulating this reply quite bluntly, but I would like to base my choice in this matter on all information available.
To replace IPC fully with by setting threshold and baseline would require electronic interrun calibration using some kind of internal instrument standard. This is very tricky, but several of the leading manufacturers claim they have improved the electronics of their isntrument and that the calibration is good enough. It is difficult to judge without detailed information and without actually testing experimentally. I presume sooner or later somebody will publish a study revealing the precision of the electronic calibration. But so far, to my knowledge, no such study has been reported.
4 posts • Page 1 of 1
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