Troubleshooting a qRT-PCR problem with conventional PCR- is this a plausibl

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jiujitsuisfun
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Troubleshooting a qRT-PCR problem with conventional PCR- is this a plausibl

Hey ladies and gentlemen!  I’m using qPCR to investigate how gene expression changes under different conditions in bacteria.  I did RNA extractions of the bacteria under the desired conditions and converted it to cDNA using the iScript cDNA synthesis protocol.  Our thermocycler malfunctioned towards the end of the reaction, so I need to verify that I actually have the cDNA I want and not just a failed reaction.  

My question is:  can I set up a conventional PCR using the cDNA as a template, and use primers for a bacterial housekeeping gene, run the PCR product on a gel and hope to verify that cDNA is present in that way?  I would prefer not to do it with qPCR if possible, because sybr green is expensive!  My concerns are  (1) that using the same 2-step protocol with the same temperatures and number of cycles won’t work because the supermix I’m using for regular PCR is slightly different than the one for qPCR, and (2) the amount of amplified DNA will be too small to visualize on a gel because ethidium bromide is way less sensitive than sybr green.  Before doing the cDNA synthesis, I normalized the RNA to 0.44 ng/µL as per the instructions, which is very small compared to the amount of template that a person would normally use for PCR.  Can I remedy this by doing a lot of cycles (like 50+) for my PCR?  

Or is this idea doomed to fail, in which case should I just bite the bullet and run qPCR on my unverified cDNA?  Thank you in advance!

Ivan Delgado
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Although you started with 0

Although you started with 0.44 ng/uL of RNA, it is likely that you got a lot more cDNA if your RT reaction worked. When you say that the thermocycler malfunctioned "towards the end of the reaction", how close to the end was the reaction? In other words, out of the total length of the cDNA synthesis reaction, at what point did the instrument malfunctioned. If the instrument failed with more than 20% of the reaction's time still unfinished then I would guess that you did not get cDNA synthesis.

If the reaction was almost done when the instrument malfunctioned then you may have some cDNA. Furthermore, if the amount of cDNA you synthesized is not limiting I would go ahead and run a test PCR. I would not go beyond 40 cycles since you start getting all sorts of non-specific artifacts beyond that many cycles.

Finally you can bite the bullet and run your qPCR without testing your cDNA. If you do this I would strongly recommend running a control reaction to makes sure everything is working as it should. Typically a good housekeeping gene is an ideal control for such reactions. 

Good luck

jiujitsuisfun
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 Wow, thanks for the quick

 Wow, thanks for the quick reply.  First, I was unaware that the cDNA concentration would be higher than the RNA concentration.  I thought that each mRNA gets degraded by RNase H after being reverse transcribed once, so that if the RT reaction was 100% efficient, we would still have 0.44ng/uL of cDNA.  I believe I may be making a wrong assumption though!

What happened was the thermocycler was programmed to hold at 4 degrees C after finishing the run, but instead it was holding at 40-something degrees instead.

My decision (for now anyway) is to do a small qPCR run to test the cDNA.  Two 25 uL reactions with primers for different housekeeping genes, one 25 uL reaction with new, untested primers for a gene I'm interested in, and one NTC with primers for one of the housekeeping genes.     

 

Ivan Delgado
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Not to play around with

Not to play around with semantics, but strictly speaking if you got an exact duplication of RNA to cDNA, then you would have 0.88 ng/uL of cDNA (since cDNA is double stranded that thus has twice the amount of nucleic acid as the same amount of RNA)

cDNA by nature is not very stable so holding your reaction at 40oC instead of 4oC will likely degrade all your cDNA in short order.

Testing your cDNA may be an OK idea, but given the very unusual treatment your cDNA has gone through my recommendation would be to start all over again.