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Q-PCR: Strange Amplification Curve shape (non exponential)


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#1 jojoziggy

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Posted 22 December 2009 - 11:14 AM

Hi all, first time poster - I made this account to ask this question, so please be kind. ;)

I am having a problem with the amplification curves in my Quantitative PCR having a very strange shape (please see attached figure).
The problem is sample specific, and is not due to the primers (on the same plate, both sets of primers worked fine in "Sample 2", and both failed in "Sample 1"). I'm just showing a subset of the samples here. About half the samples on this plate failed. I set up the reaction twice, and the same samples failed the second time as well, so I really do think it's a sample problem. The samples that failed were all from the same prep - I am thinking it's some kind of contaminant that was introduced during the prep (either in the RNA or cDNA step), but I'm not sure how to figure out what it is or how to avoid the problem in the future. Any ideas would be greatly appreciated!!

some other notes:
1. These end products of the QPCR ALL look fine (right size, proper band strength) on a gel. So it is not as if the weird ones are not amplifying.
2. I ran this on the StepPlus from Applied Biosystems, and the SYBR reagent I used was from BioRad. These are the same reagents I and everyone else in the lab has used successfully in the past.
3. The melt curves look the same for the samples whether they have "weird" or "normal" amplification curves
4. When I repeated the run, I changed the location in the plate each sample was in, so that in case it was a problem with the machine reading particular parts of the plate it should affect different samples, but the same samples were affected.
5. The samples are all fly cDNA made from RNA using Invitrogen M-MLV and oligo-dTs. I use a 2-step protocol where I purify the RNA (Trizol) then make cDNA as a second step.

Attached Thumbnails

  • ExamplePlots.jpg


#2 pDNA

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Posted 28 December 2009 - 08:48 AM

i have the same feeling that you have ...there must be something wrong with your template.

The Sample 1 control looks like if there is no template at all?

I'm not an expert for qPCR but it could be due to some PCR inhibitor in your sample.
Try to make 10-fold dilutions of your template ...maybe this effect will go away.

There is a very good yahoo group where all the gurus of qPCR are part of ...and they have helped me several times (just give it a try!)
http://tech.groups.y...qpcrlistserver/

Maybe this will help you get along!

Regards,
p

#3 jojoziggy

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Posted 03 January 2010 - 01:44 PM

i have the same feeling that you have ...there must be something wrong with your template.

The Sample 1 control looks like if there is no template at all?

I'm not an expert for qPCR but it could be due to some PCR inhibitor in your sample.
Try to make 10-fold dilutions of your template ...maybe this effect will go away.

There is a very good yahoo group where all the gurus of qPCR are part of ...and they have helped me several times (just give it a try!)
http://tech.groups.y...qpcrlistserver/

Maybe this will help you get along!

Regards,
p


Great, I'll try that group - see you there perhaps. :P I agree, I'm pretty sure it is some kind of contaminant, but I was hoping to get an idea of what contaminant it was so I could avoid messing up future samples.
I will try the dilution approach, and probably also do a phenol-chloroform extraction on a sample or two to see if that clears it up. Thanks!

#4 Dniesje

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Posted 07 January 2010 - 06:08 AM

Looking at your curves, I get the idea that you do have amplification but that the fluorescence is to low to get a good curve.
This could mean there is a problem with the setting in the software or that you don't have the right amount/concentration of probes in you tubes.

70.000k is on the low side, so try to play with the concentration of probes.
If it doesn't change, that there is a fat chance you have inhibitors.




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