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qPCR, Appearance of amplification curve for my minus RT control


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6 replies to this topic

#1 Mohamed 1984

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Posted 15 September 2012 - 04:27 PM

Iam really get shocked yesterday. The reason behind is that i run an experiment of qPCR to check the quality of hosue keeping gene. The amplification curve of the sample was perfect but i found that my minus RT control showed a normal amplification while iam sure that i did not miss add any cDNA sample to it. What may be the cause ? and how i can correct?


Thanks in advance

#2 phage434

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Posted 15 September 2012 - 07:11 PM

This may indicate that you have DNA contamination in your RNA preparation. The -RT control prevents the RNA from being converted to cDNA, but if genomic DNA is present in the RNA prep, then it will amplify.

#3 Mohamed 1984

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Posted 16 September 2012 - 08:39 AM

So, This means that the gene iam looking for is present in the genomic DNA also ? So how can i deal with such case

#4 phage434

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Posted 16 September 2012 - 10:27 AM

The RNA is a copy of the genomic DNA, so if there is genomic DNA in your RNA prep, then the gene will be present (although it may have introns splitting it into many parts).
Typically this is dealt with by treating extracted RNA with DNAse enzyme. If you gene has introns, then using primers that span the intron can also be an effective technique to differentiate cDNA (from your RNA sample) from genomic DNA contamination.

#5 Mohamed 1984

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Posted 18 September 2012 - 05:06 PM

I an not ath the point of intron . u say it again

#6 Mohamed 1984

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Posted 18 September 2012 - 05:06 PM

i do not understand the point of intron

#7 Trof

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Posted 21 September 2012 - 11:41 AM

The gene coded by DNA is actualy present there in several parts. Those parts are joined in the final (mature) mRNA and the complete mRNA is translated to protein.

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mRNA has the same sequence as DNA but misses the introns that were cut off. You designed primers for mRNA (that's complementary to cDNA), if you have both primers in the red exon for example, the primers will amplify cDNA but also the DNA if it's present and you can't tell the difference.
By designing one primer in the green exon and the other one in red, only cDNA will give you desired length, the product from DNA would be longer so you can distinguish them. If the intron between the two exons is very long (i.e. longer than say 1000-2000 bp) the DNA would not amplify at all. This is called "span the intron".

Also by designing one of the primers on the exon-exon boundary (like half of the primer in the green exon, second half in red exon) would cause that the primer won't even bind to DNA, so you will have no amplification from DNA.

These two techniques are used to prevent amplification of trace DNA in your cDNA samples. Also it's better to treat the mRNA with DNase, but that sometimes isn't 100% sure. RT- control is for checking you don't have DNA in your cDNA sample.

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