Interplate calibrators with 10 samples

[ongwaik]

Hi all,

since I have never handled so many samples before, I am confused about introducing interplate calibrators to different plates. So I have the following questions:

1. If I have 10 samples and I would like to amplify 4 genes from each sample, how do I setup the interplate calibrators? I would arrange the plate setup in such a way that plate 1 will house sample 1, 2, 3, 4 and 5 with four genes each (triplicates). Plate 2 will have sample 6, 7, 8, 9 and 10 with the same four genes each.

2. Must each interplate calibrator represent a gene? Or can I just perform IPC for one gene for two plates?

3. What about the sample? Which sample should I use for IPC from among the 10? Can I instead use a positive control as the template?

4. Finally under what circumstances can I avoid including IPC into the setup? I vaguely remember reading from somewhere that sample arrangement should be given priority instead of genes arrangement on a plate so that IPC can be avoided. Is that true? My arrangement described in 1) is giving priority to the sample, is that right?

Thank you in advance and looking forward to valuable reply soon.

Regards
Ong

[ongwaik]

Hi all,

I think I have found my answers regarding the sample arrangement. I can put all my samples into one plate if I perform a duplicate instead of a triplicate. But in situation where my samples have to be separated into different plates, how do I introduce IPCs into different plates? So question no.2 and 3 from my previous post remains.

Thanks in advance and have a nice weekend.

Regards

[Mikael Kubista]

Interplate calibrators (IPC) are used to correct for variations between runs due to instrument settings, which are primarily the threshold level and gain. Therefore one IPC per instrument channel is sufficient. Hence, if you use dye reporters, a single IPC is fine. If you use probes, one IPC per probe is needed. Some old papers use one IPC per assay, but that is not recommended. Not only is it waste of material/wells, but it is likely to introduce more noise due to random variations than the systematic noise it actually removes. The IPC should be a reliable sample that can be assayed highly reproducibly (low standard deviation on technical replicates). It can be a standard sample that you use every time.

Note, if you can use a plate setup such that all genes from each sample are measured in the same plate or each gene is always assayed in all samples in the same plate, then interplate variation cancels and calibration is not necessary. This is easily verified in GenEx.

Global interplate calibration is available in GenEx ver 5.0 and later

Good luck!