real time PCR trouble - real time PCR trouble (Aug/27/2005 )

I try to compare two genes expression by using real time PCR.
First, I extracted RNA from tissue samples.(Trizol invitrogen)
Second, I synthesized cDNA by using First strand synthesis kit
(invitrogen) and after that added DNaseI to cDNA sample(invitrogen amplification glade)
Third, I checked gene expression.(ABI 7000 systems)
But amplification curve was not good.
I usually can not see signal in low cycle, but could see as smooth amplification this time.
And I could see small plateau in middle cycle.
And in the last place, amplification signal were seen.
why?
First, I thought that RNA purity wasn't good and tried ethanol precipitation.
The results were a little better than before but amplification curve were bad.
The point I was anxious about was the amount of precipitation.
Protein or other contaminations might exist.
(Or just DNaseI?)
260nm/280nm is 1.6 and not so good after ethanol precipitation
After that, I tried silica gel PCR clean up kit (promega) for purification.
But I couldn't get good results.
I found that by diluting cDNA, amplification was good.
But I want to use concentrated cDNA samples.

Please teach me why amplification curve is not good and what should I do.
phenol/chloroform extraction might be good.
but I am anxious about loss of cDNA.

Sorry for poor English.

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you should check your RNA integrity by running it in agarose gel. For doing rt-qPCr, the total RNA must be good if there are any degradations then you should do a new prep.

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Thank you very much for replying my question.
I checked my RNA integrity by 1XMOPS 1%agarose gel and no degradations were seen.

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If you get better results with diluted samples, then maybe you should use it. Too concentrated samples can also give bad amplification plots.

What kind of detection system are you using?

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I'm using ABI SYBR green system combined with ABI 7000.
I think Taq man probe may be best but too expensive because I am going to analyze 100 genes.

After further experiment I found that there is same inclination (same amplification curve, for example small signal in low cycle)
in the case of no primer control ,but not in no template cotrol.
So primers, proteins or something may be exist in cDNA sample.
Primers(random + dT) which is used in cDNA synthesis, may cause bad amplification curve.
What do you think?
And I also I checked melting curve and found it is good.

I want to non-diluted cDNA sample because it is possible that I can't detect enough signal to analyze.
The density of non-diluted cDNA is not enough dense this time.

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hey! make sure you DNase treat your RNA not your cDNA!!!! you won't have any template in your reaction if you DNase treat in the order that you say you have!

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Did you check your primer system with genomic DNA? You could test the amplification by itself.
You could also titrate the concentrations of the primers or change the Mg2+/dNTP/buffer/SYBR concentration in your reaction.
And I don´t know what you mean with "small signal". Do you mean the fluorescence height?
Did you check the PCR pcroducts in an agarose gel?

I asked more questions, than you did, but I think the answers could help us to find a solution for your problem.

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As stated by someone above: DNAse I also digested RNA:DNA hybrids !!
So you must DNAse-treat before RT step.

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if the DNAse treatment was a typo and you are doing that correctly (to the RNA, not the cDNA) then have you optimized your primers?

I don't care how good they look with primer express, you still have to determine optimal primer concentration empirically for qPCR to work properly. Even if you do everything else right, your primers must be in the right ratios or you will not get good amplification using real-time

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did you dilute your cDNA? I had also problems with curves when I forgot to dilute it, after10x dilution I had no problems. Bear in mind that in some RT kits /I use kit by Fermentas/ is merkaptoetanol, which may denature BSA /if used/ in qRT-PCR

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