Inter-assay positional variance - Ct values seem to be dependent upon position of wells (Jul/26/2006 )

Hello,

I have been running several real time PCR plates lately, comparing relative expressions between two exons (plate is split in half). I am doing duplicates and I have 24 samples, so I'm using all 96 wells for my reactions. Recently I've been noticing a trend with my treatment that doesn't seem to be biological. Thus, I switched the positions of the samples, with the one exon on the top half and the other on the bottom half. The results reversed, following positional trends, not the treatment.

So I then ran some plates with 96 identical reactions, and noticed that the Ct values became lower for the wells closer to the center of the plate. For example, I would get Ct values of 31.8 near the center and 33 near the corner.

Bio Rad tech support told me to realign the masks (which I did), but it did not change the trends.
I am running these on a iCycler iQ with SYBR green supermix in 20 uL, by the way.

Has anyone experienced this before? Any Advice? Thanks.

I do not know that machine, but I can tell you that with ABI's machines, 1) make sure the bulbs are good 2)get the machine a check-up and 3) if you have any contaminating background fluorescence in some of the wells, it needs to be assessed and removed. hopefully BioRad can help you determine how to do this?

good luck

Run 50 mkl reactions

and see paper in Analytical Biochem

Sample volume and plate bias in real-time

[quote name='escius' date='Jul 27 2006, 03:21 AM' post='61397']
Hello,

I have been running several real time PCR plates lately, comparing relative expressions between two exons (plate is split in half). I am doing duplicates and I have 24 samples, so I'm using all 96 wells for my reactions. Recently I've been noticing a trend with my treatment that doesn't seem to be biological. Thus, I switched the positions of the samples, with the one exon on the top half and the other on the bottom half. The results reversed, following positional trends, not the treatment.

So I then ran some plates with 96 identical reactions, and noticed that the Ct values became lower for the wells closer to the center of the plate. For example, I would get Ct values of 31.8 near the center and 33 near the corner.

Bio Rad tech support told me to realign the masks (which I did), but it did not change the trends.
I am running these on a iCycler iQ with SYBR green supermix in 20 uL, by the way.

Has anyone experienced this before? Any Advice? Thanks.


This results for BioRad were published in 2004 in Anal Biochem

Their reccomendations - use 50 mkl reactions and place samples in
the center of the block
Placing samples in the cener of heat block is "OLD TRIED AND TESTED WAY"
for ALL heat block thermocyclers begining from PE 480 and finishing by modern Peltier

( the only that didnot suffer were PE9600 and 2400 because they used strongly "oversized"
blocks and your samples automatically appeared in the center of block
)

What's the efficiency of your PCR reactions? If your reactions are inefficient, or your PCR conditions are suboptimal (ie. too short annealing/extension times, or amplicons too long, etc), then your PCR reactions will be much more susceptible to the very subtle temperature varations that can occur across a thermal cycler block.

Also be sure to check for evaporation around the plate edges (if you're using film to seal the plates), as evaporation can throw off your Ct values as well.