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Heart ache in real time


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#1 Lost_in_transcription

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Posted 25 September 2009 - 10:39 AM

Hi!

I'm putting this message up as I am at a loss to explain what I am seeing and hope that someone out there can help. I am currently doing extractions from "stomached" lymph nodes (basically the liquid/ bits you get when you squish lymph nodes in buffer). I am using the Qiagen DNeasy blood and tissue kit for the extraction and using PBS on its own as an extraction control. I am then running these extractions using Taqman real time PCR for 40 cycles using a 6-FAM 5' labelled 3' Black hole quencher probe. I have noticed that whilst there is no/ very poor amplification of my bacterial target sequence (this material is culture positive) in my samples, there is a signal (around ct 27 at strongest but normally around 33-35 cycles) in my extraction control. I have an inhibition control PCR (18SrRNA) for each sample, which is run in a separate well and this is always positive (at <18 cycles), so my question is, could it be the case that the "host DNA" is inhibiting the amplification of my bacterial target and if so, what can I do (I'm assuming that a mere dilution will just make my bacterial signal even quicker)? I know that there are columns that reduce eukaryotic DNA but is there any other method available that will do the same job without too much manipulation? Any suggestions will be considered!
The other major problem is the amplification in the extraction control itself. We have checked all that we can (namely the PBS) to see if this generates a signal on its own but we consistantly get no signal. Yet after running through the column, we see something. The columns are one use only and are individually sealed. My boss has suggested that there may be a chemical interaction involving the buffer salts and the probe. Does anybody know if this is possible as the curve generated looks real. Many thanks in advance and have a great day!

#2 Unagi

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Posted 28 September 2009 - 02:30 AM

If the probe is being chemically hydrolised, then I would expect to see a gradual increase in signal, but not an exponential one, like seen in a genuine amplification curve.

Is your bacterial target ubiquitous? eg: 16s? Such assays are notoriously difficult to keep clean, as free 16s DNA is virutually everywhere, including the polymerase. Perhaps this is why you are picking up a signal in your PBS after column extraction. Of course, another possibility is that your prep area, or something in it has been contaminated by the assay cDNA or a plasmid containing the target sequence, which would fit with the neg control coming up at ~ Ct 35.

You could try a a small set of serial dilutions to see if that helps with any unforseen inhibition, either due to some contaminant or host DNA.

Another possibility is that the primers are interacting with host DNA and being locked out of the reaction, or simply that the oligo sequences do not match the target bacterial sequence.

Hi!

I'm putting this message up as I am at a loss to explain what I am seeing and hope that someone out there can help. I am currently doing extractions from "stomached" lymph nodes (basically the liquid/ bits you get when you squish lymph nodes in buffer). I am using the Qiagen DNeasy blood and tissue kit for the extraction and using PBS on its own as an extraction control. I am then running these extractions using Taqman real time PCR for 40 cycles using a 6-FAM 5' labelled 3' Black hole quencher probe. I have noticed that whilst there is no/ very poor amplification of my bacterial target sequence (this material is culture positive) in my samples, there is a signal (around ct 27 at strongest but normally around 33-35 cycles) in my extraction control. I have an inhibition control PCR (18SrRNA) for each sample, which is run in a separate well and this is always positive (at <18 cycles), so my question is, could it be the case that the "host DNA" is inhibiting the amplification of my bacterial target and if so, what can I do (I'm assuming that a mere dilution will just make my bacterial signal even quicker)? I know that there are columns that reduce eukaryotic DNA but is there any other method available that will do the same job without too much manipulation? Any suggestions will be considered!
The other major problem is the amplification in the extraction control itself. We have checked all that we can (namely the PBS) to see if this generates a signal on its own but we consistantly get no signal. Yet after running through the column, we see something. The columns are one use only and are individually sealed. My boss has suggested that there may be a chemical interaction involving the buffer salts and the probe. Does anybody know if this is possible as the curve generated looks real. Many thanks in advance and have a great day!






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