qPCR RT- controls VS RNA

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qPCR RT- controls VS RNA

Postby mouton2003 » Mar 03 2009 11:01 am

I am new in qPCR field. I wonder if we test RNA on qPCR. We got no amplification or at least 10 Ct difference with our cDNA specific signal whose primers were designed with small intron and can amplify genomic DNA under the same reaction condition. Could we get conclusion that we do not have problem of genomic DNA contamination. Is this better than we do RT - comparing with RT+ . Because if the RNA contaminated, then the RT- should be contaminated also.

Moreover, even we try to design trans-exonic primers, there are still some genes without intron. RT- could not guarantee that we amplify only cDNA but not genomic DNA for this kind of genes. am I right for this concern?

Do someone have experience for rat liver tissue oligo efficiency test. which amount of template we should start. I know that liver is RNA rich tissue, if we use too much, it will inhibit the reaction.

Thank you very much in advance
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Postby mchlbrmn » Mar 03 2009 5:51 pm

I don't quite understand. An RT- control is the same as an RNA control, except that the RT- also controls for any contamination from, or affects on PCR from the RT buffer, dNTPs, etc.

I always put the same amount of RNA / well, not a number of cells/ well.

I sometimes run a well with some genomic DNA that I have so I can see if it can be amplified by my primers, and the melt peak of the product.
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Postby mouton2003 » Mar 05 2009 6:43 am

Thank you very much for your reply.

mchlbrmn wrote:I don't quite understand. An RT- control is the same as an RNA control, except that the RT- also controls for any contamination from, or affects on PCR from the RT buffer, dNTPs, etc. .


You are right. but if in the RNA, you have genomic contamination, it will be taken into both RT+ and RT-. do you think that RNA qPCR can tell us whether we have contamination probelm? whether we could go further for cDNA synthesis directly or being treated again by DNase before cDNA synthesis.



mchlbrmn wrote:I sometimes run a well with some genomic DNA that I have so I can see if it can be amplified by my primers, and the melt peak of the product.


We run also genomic DNA, while sometimes for some oligos, we could not distinguish the melt peak from genomic to specific signal. do we need to make new primers?

thanks a lot.
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Postby mchlbrmn » Mar 05 2009 3:47 pm

I think DNAse treatment can be imperfect, and traces of DNA may survive. Repeating the treatment may not solve the problem.
If you observe a different melt peak for genomic DNA, good.
It seems to me that RT- control does the same thing as an RNA control, but is an even better control because the PCR conditions are more identical, and it checks for contamination of more reagents that make up the experiment.
Even with a tiny amount of genomic contamination, the experiment can still be good. I agree with you that if the genomic DNA product only shows up with a Ct much higher than your smallest experimental sample, then it is not really affecting your experiment.
Moreover, even we try to design trans-exonic primers, there are still some genes without intron. RT- could not guarantee that we amplify only cDNA but not genomic DNA for this kind of genes. am I right for this concern?

For genes with or without introns an RT- control should be negative. This tells you that any product from the RT+ is cDNA, correct? If a small amount of genomic DNA is present, or another artifact band appears, the RT- can tell you if the amount is so small that it doesn't matter.

Sometimes artifacts or nonspecific problems also can affect the results. Since these cannot be perfectly predicted for new primers, it may be a good idea to design two primers for each gene, and use the one that works the best.

I am not familiar with the rat liver test.

Gtood luck sheep.
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You have no problem

Postby JMG » Mar 07 2009 12:54 pm

HelloMouton2003

If your -RT signal (gDNA) is 10 Ct away from your
+RT rxn Ct, you have no problem.


At 80% efficiency:
If your gDNA Ct (-RT rxn) is 6 cycles away from
your RNA (+gDNA contamination) Ct, the additional
(gDNA) contribution to your RNA Ct is only ~3% of
your total Ct signal. A difference of 10 Cts is only
about a 0.3% contribution. So you have no worry.
At 100% efficiency, the contribution values are even
smaller since the same Ct distances mean greater
fold differences, etc.

Sounds like your DNase treatments are very good.

Regards,

JMG
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qPCR for rat liver RNA

Postby JMG » Mar 07 2009 1:11 pm

Mouse liver RNA extracts work great at final analyzed
concentrations of 0.6 ng total RNA/uL in well for qPCR.

The standard curves are good in a range from:

1.5 ng/uL to 0.15 ng/uL for all targets we tested.
So make your serial dilutions of standard curve
material within that range from 1.5 to 0.15 ng/uL.

Rat liver should be similar to mouse liver.

One does not always need a large dynamic range for
standard curves. As long as the dynamic range you're
working in demonstrates high amplification efficiency -
and as long as your individual sample concentrations
all fall within that curve range and are all diluted to the
same ng/uL by the time they exist in-well (0.6 ng/uL),
and as long as each isolate, by the time it exists in-well
has incurred at least a 1:100 overall dilution by the time
it is analyzed at 0.6 ng/uL in-well (to insure diluting out
of inhibitory material), you will be golden.

I just assisted the development of a successful qPCR
for mouse liver RNA (One-Step qPCR) a week ago --
and these parameters worked like a charm.

Hope this helps.

JMG

PS your samples may be able to be used within a
more concentrated overall dynamic range -- but try
these parameters first -- they will waste the least
amount of sample.
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Postby mouton2003 » Mar 16 2009 8:23 am

Thanks a lot for all of you. good luck for your work.
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