IPC variability

Here we discuss the use and processing of inter-plate calibrators

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IPC variability

Postby Curious Georg » Fri Aug 16, 2013 8:46 am

How much variability in the IPC is acceptable?
Curious Georg
 
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Joined: Fri Aug 16, 2013 8:37 am

Re: IPC variability

Postby Mikael Kubista » Fri Aug 16, 2013 8:48 am

Typically, we express IP variability in cycles (SD) rather
than relative SD, because it should be independent of Cq. But that is less
important.

Assuming you used IPC’s, how many replicated per plate did you run? We
recommend minimum of triplicates, but it depends on the precision you need
and, of course, how good your IPC is.

Have you tested the performance of your IPC? It should be tested for
repeatability (intra-run precision) by running full plate of replicates and
should have an SD < 0.1 cycles on a well performing qPCR instrument. In
fact, this test reflects the performance of the instrument as well as of the
assay and not all instruments live up to this. In fact, most manufacturers
only guarantee SD < 0.25 cycles.

What Cq does your IPC have? Should typically be 20-25 cycles, depending on
instrument.

Most importantly, have you tested the long term reproducibility (inter-run
precision) of your IPC? It should be stable over the time period of your
experiment. It is critical the IPC can be stored over time, so changes are
not introduced. The TATAA IPC is provided in small aliquots to avoid
freeze-thawing, which is known to introduce variability, and in stabilizing
media:

<http://www.tataa.com/products-page/quality-control/>
http://www.tataa.com/products-page/quality-control/

In summary, the interplate calibration performance depends on the factors:
1) the performance of your IPC, the performance of your instrument, the
number of IPC replicates you run. If you want to read more about IPC check:
www,qpcrforum.com and the manual for the TATAA IPC. Once you performed your
experiment it’s easy to correct for inter-plate variation from the IPC data.
You can do it manually or use the automated workflow in software such as
GenEx.

I should mention the master mix may compromise your data, but I presume you
have validated you are using a good one (avoiding the cheapest on the
market).

Good luck!
Mikael Kubista
 
Posts: 455
Joined: Tue Jul 01, 2008 12:28 pm


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