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weird QPCR curves


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#1 uglearner

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Posted 14 February 2011 - 02:13 PM

Hi ,

I have been trying to run viral RNA using the Biorad one step RT-PCR kit for probes. I am using Cy5 as the probe for this virus. I have also done the QPCR with SYBR green which works quite well( picture attached) but for some reason the run with the Cy5 probe doesn't work at all. I thought that my primers / concn wasn't correct but how can the reaction work with the SYBR green kit and not with Cy5. Could someone kindly interpret these curves for me and help me troubleshoot. Thanks.

The first image is for the RNA sample that has been run with SYBR green mix and also the Biorad one step PCR kit. As you can see the SYBR green works better than the Cy5. sybrgreenpcr.png

The second image is that of qPCR run with Biorad one step PCR kit alone. The nanodrop concentration of my RNA was 118 ng, I used -1,-2 and -3 dilutions for this run.qpcr hav.png

#2 UBClabbie

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Posted 17 February 2011 - 04:39 PM

Do you mean you are using Taqman and a probe for Cy5 and it isn't working? Maybe the formulation of your reaction is faulty or your mastermix doesn't work well with that dye... Did you run Taqman and Sybr green in the same qPCR run? Both techniques use different methods so you can't do this.

It might be that the one step RT-qPCR kit you are using just isn't very good at all. I know it saves time to do it in one step, but the efficiency of your reaction and reliability is probably going to be affected.

#3 Trof

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Posted 21 February 2011 - 02:14 AM

biotechgirl said the first think I had in mind (there are differences in the program for SYBR and for probe - different chemistry, different fluorescent channels, different times, different point of fluorescence acquisition, check all of these), I will just add, that in my experience SYBR has always higher fluorescence than any probe, it's quite logical, there is lots of it in the product. Other experience is that fluorescent dyes other than FAM and VIC have a profoundly lower fluorescence. So you can't say just from the level of flourescence which works better.
On the other hand from the second image is obvious that something is wrong with your probe reaction. What caught my eye is the part on the beginning, where the florescence lowers, and the graph doesn't seem to reflect the quantity as SYBR does, you're probably seeing some artefacts than amplification (is it a raw graph of after some normalisation?). Try to run the product on gel to see if it amplifies at all.

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