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about real time qPCR


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#1 huotoad

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Posted 21 February 2009 - 03:55 PM

I am trying to do real time qPCR. In our lab, they usually run the PCR in a
machine, say, ABI 7300 and get the raw data, especially, the cycles and
corresponding delta R0, then they input these data into DART-PCR, a excel-
based software, and get some results. How reliable will the results produced
from DART-PCR? Another question is if we need to do quantification after
cDNA synthesis, because I found although I added the same amount of RNA, the
cDNA production sometime varies a lot. I did quantification using Nanodrop.
I am working on plants, I use 18S, UBQ and Actin for endogenous controls, I got that 18s rRNA normally does not degrade as other mRNA. When I run the three controls, I found my target gene, UBQ and Actin have high Ct value (Say 33 at one concentration), but for 18S are very very low (Ct=17) at the same concentration. Will the result suggest that my RNA degraded a lot when I prepared for cDNA?

Any suggestion will be appreciated.

#2 Trof

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Posted 22 February 2009 - 05:35 AM

I am trying to do real time qPCR. In our lab, they usually run the PCR in a
machine, say, ABI 7300 and get the raw data, especially, the cycles and
corresponding delta R0, then they input these data into DART-PCR, a excel-
based software, and get some results. How reliable will the results produced
from DART-PCR? Another question is if we need to do quantification after
cDNA synthesis, because I found although I added the same amount of RNA, the
cDNA production sometime varies a lot. I did quantification using Nanodrop.
I am working on plants, I use 18S, UBQ and Actin for endogenous controls, I got that 18s rRNA normally does not degrade as other mRNA. When I run the three controls, I found my target gene, UBQ and Actin have high Ct value (Say 33 at one concentration), but for 18S are very very low (Ct=17) at the same concentration. Will the result suggest that my RNA degraded a lot when I prepared for cDNA?

Any suggestion will be appreciated.


I don't know DART-PCR but usualy best way to analyse raw data is in the machine. ABI is a very good one to do it. I'd rely on that way more than some excell macro.

Here, we usually assume that reverse-transcription efficiency is the same in all samples, as cDNA concentrations is IMHO very hard to measure, just put the same amount of RNA to RT and do everyting the same way.

With 18S it's not about mRNA degradation. 18S is a very abundant gene and therefore not a good control gene for many targets. I recoment choosing a different control, with Ct values similar to the target genes. And generaly to use more tamplate to your reactions as Ct 33 is very high to make a correct quantification from it.

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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