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Problem with UPL real-time PCR - please help !!!


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#1 zoyd81

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Posted 16 November 2012 - 09:58 PM

Hi ;)

We are working on gene expression of two opioid receptors in human skin: OPRM1 and OPRK1 using universal probe library, UPL probes from Roche company. We do not have any problems with OPRK1 becouse it.s gowing fine on all probes, but problems starts with OPRM1. We detect by Probe Finder three different primer pairs, which react with constitutive regions for OPRM1. In the begining we make real-time but using Sybr Green. For positive control we use cDNA from human brain. All primers react fine, in the brain and in the skin. Melting curve also look good.Sequencioning results give us the same sequences in the brain, skin as reference sequence in NCBI. The same reaction we do on the same material: brain and skin cDNA but using UPL probes. All primers and UPL probes detect expression of OPRM1 in the brain, but in the skin not !!!
We have the same problem with OPRM1 when we use ready to use gene expression assays from Applied Biosystem. In brain everything was ok, but in the skin we do not have any expression.
We also make this experiments using very good cDNA material from the keratinocyte cultures. OPRM1 work with sybr but not with UPL probes.

Please HELP

#2 Trof

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Posted 17 November 2012 - 10:20 AM

The SYBR works on both samples, sequence is the same for bothn, but probes doesn't want to bind in skin sample... hmm. DId you run the other sucesfull OPRK1 UPL assay on the exact same cDNA? If so, the problem is not in the sample. It isn't probably also in the probe, since that works in the other sample.
Just assuming there isn't any common mistake (like forgotting the probe in the other mix or so) and that this happens repeatedly.

I would run the SYBR and probe reaction on skin and brain side by side and put them on gel, that really the probe reaction is amplifing.
Then I would isolate from gel the probe reaction amplicon from skin (if it's positive) and sequence this exact amplicon and particulary look into the region where the UPL probe/ABI probe should be binding. Manually.

If still no explanation is found and you need the results, just use SYBR then.

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