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What could be the possible reasons for a RT-PCR experiment that was working fine


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#1 Sakumi

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Posted 19 September 2015 - 06:54 PM

What could be the possible reasons for a RT-PCR experiment that was working fine to stop working, if nothing was changed? I seem to see quite a few questions about this. And I've been battling with this problem for quite a few months now....

 

Thank you!

 

Sakumi

 

 



#2 bob1

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Posted 20 September 2015 - 12:52 PM

The list could be as long as your arm...

 

RNA degradation,

DNase degradation

reverse transcripase not working

DNA degradation

Primer degratation

polymerase not working

detection system not working

buffers used wrongly somewhere

 

Details would help - what RT are you doing - one step? two step? is the qPCR or just regular PCR?



#3 Sakumi

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Posted 21 September 2015 - 06:00 AM

I'm doing two step end point RT-PCR. So I extract total RNA using Trizol, synthesize cDNA, then do PCR and run the products out on a gel.

 

It seems strange because I didn't change anything....



#4 Mad Researcher

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Posted 23 September 2015 - 08:36 AM

Maybe try with new mastermix, water and if you still have RNA, try synthesizing new cDNA from the RNA stock and try.

 

And try changing one thing at a time to know where the problem was. This should help.


Cheers,

Mad Researcher

#5 phage434

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Posted 23 September 2015 - 10:19 AM

If you have DNA from a previous run, then you can determine if the problem is in the RNA & RT step, or in the DNA amplification step.  Usually the problem is in the RNA part of the protocol.






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