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A problem with PCR array


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#1 dnasequencer

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Posted 12 December 2014 - 06:06 AM

Hi,

I have used PCR array from qiagen to test genes expressed in cancer cells, but have got no amplification at all.

I have checked my RNA concentration, integrity and the cDNA I used and they were all fine. So what do you think this problem is due to?



#2 CPRES

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Posted 28 January 2015 - 04:23 AM

This is too general a question. There could be one or two of about 74 things that could be going wrong. See here for some PCR array manuals and troubleshooting guides, think critically, get help from a senior colleague to get an objective assessment of your techniques that you may be missing.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#3 Trof

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Posted 28 January 2015 - 05:53 AM

Does the array have some internal controls? Or some reference genes, that should be always present?

 

By the way, how did you check that the cDNA is fine?


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

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#4 dnasequencer

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Posted 28 January 2015 - 08:56 AM

Yes the arrays have internal controls and but there is no amplification I am getting cracked lines instead of the exponential amplification curves.

I checked my cDNA using taqman primers: actin, GAPDH and vimentin. And it turned to be that the problem with the PCR plates themselves.



#5 Trof

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Posted 28 January 2015 - 12:46 PM

I see, so your cDNA amplifies and the internal controls don't.

 

Just one idea, check that you have a correct setting for a detector (fluorescent channel), in some cyclers (ABI) if the program is run with the wrong detector, the data is accessible once you select the right one. This might save your run if that's the case.

 

Otherwise you lost your samples for now. Next step would be rechecking that you followed the manual from the array and didn't forget anything. If you are sure about you did, and that the storrage was appropriate, then you can only contact Qiagen to tell them. They may have some ideas too, or they will decide the product was bad send you a new one.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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