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qPCR of Intronless gene


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#1 Rifa

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Posted 31 October 2013 - 01:55 PM

I am working on some intronless genes. So I couldn't designed exon-intron spanning primers. I treated RNAse free DNase 37 degree for 15 min before cDNA synthesis. But my negative RT control showing amplifications i.e. positive signal after 29/30 cycles. I did qPCR twice, every time it is showing positive signal in no RT control even after DNase treatment. Does that mean my Dnase treatment is not enough and there are traces of genomic DNA contamination in my samples? But I can not design exon-intron spanning primers as I am working on intronless genes. 

Can anyone help me anyway or any suggestions?



#2 bob1

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Posted 31 October 2013 - 02:13 PM

Amplifications at 30+ indicate that there are probably still several copies of the DNA present - longer DNase treatment should work.   If you had said 35+ I would have said that there may be one or two - as by this point the number of copies generated by the PCR are immense even for a single stretch of DNA.

 

I'm hesitant to say it as I'm not sure that it is a usable/viable technique for this sort of thing, but you could potentially cross-link or degrade (UV?) the DNA so that the strands can't separate and then perform the RNA extraction.  The risk would be that most agents that can be used to cross-link DNA would also affect RNA.



#3 doxorubicin

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Posted 01 November 2013 - 12:50 AM

I've personally had some good luck with Agilent's Total RNA Isolation Mini Kit to purify only RNA.  I used this when transfecting mammalian expression constructs followed by qPCR.  You can DNAse treat your RNA in solution and just clean up with this kit and it should be fine.  On a related note, our alternative careers group has written a detailed protocol for designing qPCR primers here: https://sites.google...wn-qpcr-primers



#4 Z.Onur

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Posted 01 November 2013 - 07:00 AM

Could your equipments such as pipets (especially) or solutions, kits be contaminated with your sample? Do the other people in your lab have the same problem or do you face with the same problem while performing qPCR with different genes.

#5 Trof

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Posted 20 November 2013 - 05:49 PM

We use Trizol RNA isolation and Ambion's TURBO DNA-free Kit for DNase treatment, even for non-intron spanning real-time assays, and we get no signal on RT- over 45 cycles. 

Sometimes when using on-column DNase tretments (QIAGEN columns and separately purchased QIAGEN DNase) we got an occasional contamination in RT-, but TURBO DNA-free can be used on any already isolated RNA, from column on not. Sometimes to be sure, we used both the on-column and kit DNase, since of course the ability of kit DNase to digest DNA is not unlimited. But generaly Trizol isolation is in only DNA-depleting and the kit is still sufficient for digesting the remaining DNA.

Of course if the isolation procedure ends with too much DNA, no DNase can digest it completely. So the RNA isolation should be at least somehow specific for RNA only.


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