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Is some amount of RNA obligatory for qRT-PCR?


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#1 kutayozturk

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Posted 04 April 2012 - 06:10 AM

Hi,

I wonder that is it mandatory to have same amount of RNA to use in cDNA synthesis and then qRT-PCR? If it is so, what is the purpose of the normalization primers, only to fix minor deviations caused by pipetting etc.?

#2 pcrman

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Posted 11 April 2012 - 08:01 PM

Yes, you better use very consistent amount of RNA for RT reaction across your samples. If there is a big difference in starting RNA, normalizing to internal control gene cannot sufficiently correct the difference.

#3 kutayozturk

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Posted 12 April 2012 - 02:50 AM

Thanks for your answer pcrman. I have adjusted the amount of my cDNAs to the same level, and actually running a qRT-PCR reaction at the moment. I have one more question then: Since I kept the amount of my cDNAs very low, the Ct values for housekeeping genes are around 30. The ones with less expression shows Ct at even 34-35th cycle. Now, I increased the amount of my cDNAs as much as possible. So, if I see Ct values at around 25, may I count such a result dependable?

#4 Trof

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Posted 13 April 2012 - 01:57 PM

I'm not sure what you mean by 'dependable', but to get Ct 25 from previous 35 would mean increasing the amount of cDNA more than 1000x times. I doubt you can achieve that.

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#5 kutayozturk

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Posted 17 April 2012 - 03:51 AM

I meant reliable, wrong choice of words. I prepared very dilute forms of RNAs during DNase treatment, and then I diluted them even more during cDNA synthesis. But the main question I want to ask is that if I obtain Ct values around 30th-32nd cycles, can I trust any data obtained from such a qRT-PCR experiment?

In fact I have another question, my supervisor advises me to have Ct values at the same region for all samples. In order to achieve that, I have to add 2.5 more cDNA for my gene of interest analysis than that of housekeeping genes. By doing that, I am ignoring the first point, "add equal amount of RNA". I added the pictures of the three last qRT-PCR experiments. Can you tell me if I can trust these results or not?

http://imageshack.us/g/220/9nisan.png/

edit: 1st picture is taken by using the same amount of cDNAs

Edited by kutayozturk, 17 April 2012 - 03:59 AM.


#6 Trof

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Posted 18 April 2012 - 06:45 AM

Generaly 15-30 is considered a range for a reliable quantification.
But having Ct 32 and good reproducibility of replicates.. question is, if you have such diluted RNA, you may even try to concentrate it more if you have enough of it.
Ct's for housekeeping genes and gene of interest should be similar, but I would interpret that as not having a 10 cycle difference for example. And of course gene of interest can vary, so sometimes you can't have same Ct's for all range. Generaly it's recomended to have a reference gene of similar abundance. Definitely you should add same amount of cDNA to housekeeping and to gene of interest assay even if that means a bit difference between them.

As for the picture, from the first picture, the values between 25 and 30 (green) are usable, the others.. not so much, I would say. Especially if you can add more RNA, I would do 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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