Designing the primers for qRT PCR - Query - (Sep/05/2008 )

I am trying to look the expression of ghrelin on monocytes using q RT-PCR. I am going to use the c DNA as my template. I have been asked to design exon spanning primers . so that, I can get rid of any genomic contamination. But I was just wondering, cant I get rid of genomic DNA if I design the primers in just one exon(within one exon). This way also designed primers wont amplify the introns.

Please reply asap.
Thanks in advance.

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The purpose of having two primers span introns is to let you know whther your RNA has DNA contamination or not. This is useful for regular RT-PCR because if there is DNA contamination, you may see an extra bigger band amplified from DNA template. For real-time PCR, this practice is not very helpful because any amplification from DNA or RNA will give similar signal. If two primers are in the same exon, they also amplify genomic DNA if there is DNA contamination. So in order to get correct expression data from real-time PCR, it is very important to start with DNA-free RNA.

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This is true, but your qPCR product will consist of DNA amplified from your cDNA (correct) plus product amplified from any contaminating genomc DNA present (incorrect), both of which will be the same size. Since you're measuring expression, I assume by comparing several samples, you could assume that any contaminating genomic DNA is constant between samples while the cDNA amount fluctuates, so your results would still be meaningful, but this is not an especially safe assumption, and you'll have trouble publishing results from such an experimental design.

It's much better to design your primers correctly up front, so that they each span an exon-exon junction. This way you can be assured that the only product seen is being amplified from your cDNA template.

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But if even if i get the amplified product from the genomic DNA, it should not matter because the primer still ill just amplify exons which is resposible for expression.Sorry if i have asked something very silly

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To put clear words


I am trying to look the expression of preproghrelin on monocytes using q RT-PCR. I am going to use the cDNA as my template. I have been asked to design exon spanning primers through which I can get rid of any genomic contamination. But I was just wondering, cant I get rid of genomic DNA if I design the primers in just one exon(within one exon)?. This way also designed primers wont amplify the introns.

Moreover if I'm designing a exon spanning primers with large intron(1000bp) i would not get any amplification if my cDNA is contaminated with genomic DNA rather if i just design the primers in just one exon i will definitely get the amplification from genomic DNA and the cDNA.

I have one more doubts ,if my cDNA samples are contaminated with genomic DNA and i designed primers in just one exon.How many bands will i get in the agarose gel? Why we always prefer to use cDNA as template when we are trying to look the expression of genes?

If i get the amplified product from the genomic DNA, what is the problem in that, because the primer still ill just amplify exons which is responsible for expression.Sorry if i have asked something very silly

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You've said two different things here -- in the first paragraph, you said you wanted to design primers to a single exon. In the second paragraph, you say you want to design primers to a single exon, but one which has an intron between it. The second paragraph makes no sense -- if there's an intron there, between the primer binding sites, then you're designing primers to two different exons -- there's no such thing as a single exon that contains an intron; the intron separates two exons.

Assume E and e indicate exons, and I indicates an intron. If the genomic DNA is arranged thusly:

eeeeeeeeIIIIIIIIIIIIEEEEEEEEEEEEIIIIIIIIIIIIeeeeeeeeeeeeIIIIIIIIIIIIEEEEEEEEEEE

the cDNA will be:

eeeeeeeeEEEEEEEEEEEEeeeeeeeeeeeeEEEEEEEEEEEEEE

If you design primers within a single exon (say the first eeeeeeee), they will amplify from both genomic DNA and cDNA.

If you design primers whereby each primer is wholly contained within a single exon, but each primer is to a different exon -- say a forward primer that begins at the beginning of the first exon (eeeeeeee) and a reverse primer that begins at the end of the second exon (EEEEEEEEEEEE) -- this set will amplify a product from the cDNA (of size eeeeeeeeEEEEEEEEEEEE) and will also produce a product from the genomic DNA (of size eeeeeeeeIIIIIIIIIIIIEEEEEEEEEEEE).

If you design primers such that each spans two exons (say a forward primer spanning the end of the first exon and and the start of the second exon, thus eeeeEEEEE, and a reverse primer that spans the end of the second exon and the start of the third exon, thus EEEEeeeee), these primers will only amplify from the cDNA, because the sequences eeeeEEEEE and EEEEeeeee do not exist as contiguous segments in the genomic DNA.

Only in the last case is there no possibility of amplifying genomic DNA; this is why you've been asked to design primers in this manner. The first two primer design strategies allow the possibility of genomic DNA amplification, the third design does not.



If both your primers target a single exon, you will get one band on an agarose gel, because the product from the cDNA and the product from the genomic DNA will both be exactly the same size and sequence; these products will be indistinguishable from one another (this is the first design strategy above).



Because the gene copy number on the chromosome (thus the number of DNA copies of the gene carried in the genomic DNA) stays the same regardless of conditions. However, the expression level of the gene (i.e. the number of times it is transcribed in a given amount of time) might change due to changing conditions.

What you're really attempting to measure using cDNA is the number of mRNA molecules that were created by transcription of the gene under various conditions for a given period of time. The way to do this is to first make a DNA copy of the mRNA using reverse transcriptase (this is your cDNA), and measure the number of cDNA molecules (by, for example, qPCR) to get an idea of the number of mRNA molecules that were present. Whether or not the number of mRNA molecules (measured indirectly by measuring the amount of cDNA molecules derived from them) actually equates to the number of active proteins translated from the mRNA is a matter of debate, but most people assume it does.



Because you're trying to measure the expression level of the gene (i.e. the number of mRNA molecules that were present under a given condition, as explained above), and not just the presence of the gene itself. Therefore, any product that is produced by PCR from a template not derived from an mRNA molecule is a contaminating product which will throw your results off.



Nothing to apologize for -- it's good to ask the question until you fully understand the answer. Hope this helps!

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thanks for the information

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My RNA samples are contaminated with genomic DNA and as iam using exon spanning primers ,do i really need to treat the RNA samples with DNase ?

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The exon-spanning qPCR primers will not amplify a product from the genomic DNA, but what about the RT reaction? Will the genomic DNA interfere with it, or alter the cDNA results you get? If you're using SYBR Green dye in your qPCR reaction (or any intercalating dye), the dsDNA that is present as genomic DNA will fluoresce as well as your dsDNA derived from your PCR products...

So, in general, yes -- it's always a good idea to remove as much contaminating and potentially interfering substances as is possible and practical from any reaction designed to measure something.

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But as iam using exon spanning primers ,syber green will never bind to genomic DNA becoz genomic DNA is not going to amplify to give dsDNA with the primers iam using.Dont you think so?

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