With the understanding on the advantage of using primers crossing exon-intron junctions, as correctly pointed out by Panduranga, I would put more weight on consideration of primer efficiency and specificity. If your primers designed in one exon are very efficient and specific, I would not go for a less efficient and/or specific primers just to cover exon-intron junctions. Usually you need to digest DNA in your RNA extraction anyway, because only when you do that can you accurately measure the quantity of RNA in your samples to begin with. To make sure DNA does not mess up your RT-PCR results, run a no-RT control (i.e. use equivalent amount of RNA directly as a template in PCR reaction). 

Hi Farzane,

Amplification from genomic DNA is one of the major problem you need to deal with when you are trying to amplify from single exon.  Try to include primers spanning over different exons. Otherwise you may need to treat your sample with DNAse and inactivate DNAse before going for cDNA synthesis. It requires inclusion of more number of controls to rule out amplification from genomic DNA rather than cDNA.

With the understanding on the advantage of using primers crossing exon-intron junctions, as correctly pointed out by Panduranga, I would put more weight on consideration of primer efficiency and specificity. If your primers designed in one exon are very efficient and specific, I would not go for a less efficient and/or specific primers just to cover exon-intron junctions. Usually you need to digest DNA in your RNA extraction anyway, because only when you do that can you accurately measure the quantity of RNA in your samples to begin with. To make sure DNA does not mess up your RT-PCR results, run a no-RT control (i.e. use equivalent amount of RNA directly as a template in PCR reaction). 

Hello Farzane

The best strategy in gene expression assay is exon-exon junction primer designing, but the other way is designing primer on different exons if the intron between them be long. I think because of previous experience the second way is more practical than the first one and you can get better PCR product.

Your primer MUST span an exon-exon junction; if not you can get amplification/noise from DNA in your prep. The amplicon should be short - 200 bp (less if possible), or your qPCR will be subject to PCR artifacts. And choosing a control gene to compare to may be trickier than you think. Do you have access to a droplet digital PCR? The more I do qPCR the less I like it (so complicated).

Preferably your primers should span an exon-exon boundary, or each primer should be located in a different exon (but then the intron should be large enough not to interfere with the PCR and not result in a double melting peak).

But when you have only one exon (or other circumstances where intronspanning is not possible), then you have no other option. Then just go ahead with it and include a few controls for gDNA contamination: check your RNA on agarose gel, include a no-RT control in your Reverse Transcription reaction (you could also add some extra PCRs with your primers on RNA, which should be negative). When these checks are negative then you did all you could do.

When your gene is highly or moderately expressed, then the copy number of your target in cDNA will be much higher than the target in eventual left-over gDNA that it will have no significant influence on your Ct-values.

I design primers a lot and in about 10-20% of all designs the primers are not intron spanning because I have no other option. All these primers work fine and produced good results.

Marco, thanks. Of course not all genes have introns. Your answer is great. But when a gene has an intron, I say span it if possible...in my hands it has almost always been possible.

Yes Lori, that is correct. But under some circumstances it is just not possible, and not only because the gene does not have introns. For example, some genes have a lot of splicevariants (or a few with an unfavorable splicestructure), and when you want a primer set in this case that detects all variants, or you just want to detect one splicevariant or a few, then sometimes there is no splicesite that gives you this opportunity. So then you are forced to put the primers in a single exon or the UTR.

Marco makes several good points.  If your RNA is very pure (very low level of genomic DNA contamination) and the expression level of your gene is relatively high, then it is not essential to have intron-spanning primers, although it is generally desirable where possible.  Also note that intron-spanning primers will not always prevent interference by genomic contamination because many genes, especially highly expressed ones, have processed pseudogenes inserted in the genome. An important factor is the method of RNA purification.  Column methods (like Qiagen) often leave a relatively high level of DNA contamination and the "on-column" DNase approach is, in my hands, not a very effective way to eliminate it.  In our lab, the classic Trizol approach yields RNA that is relatively free of DNA.  So further DNase treatment is only necessary for cases where the mRNA expression is low and there is potential genomic interference due to the use of intra-exon primers or the presence of a processed pseudogene.  Finally, if you are working on mouse or human, you can find many pre-designed primers (intron-spanning) at the genome browser gateway: http://genome.ucsc.edu/index.html. You should always order at least two different, unrelated pairs and test them for best performance.

Dear all

thanks a lot for your helpful comments and friendly advise

In response to Farzane R. question:

you adviced me to use one site for finding appropriate primers in rodents (http://genome.ucsc.edu/index.html) . Would you help me to find how I can search my gene primer in this site? I am looking insulin 1(ins1) primer

The UCSC site provides "pre-designed" RT-qPCR primers for human and mouse genes based on the approach described here:

http://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=444670002_7oYDLYOeCaZkxvfMUsunGaaQaSbO&c=chr11&g=qPcrPrimers

As an example, go to:

http://genome.ucsc.edu/cgi-bin/hgTracks?db=mm10&position=chr11%3A59542686-59566956&hgsid=444670002_7oYDLYOeCaZkxvfMUsunGaaQaSbO

Scroll down to near the bottom of the page where you can make selections of what to display--look at the section titled "Expression and Regulation".

You will see under there an option marked "qPCR primers". With the pull down menu, select "pack" or "full". This will cause the qPCR primer track to appear in the display above after you click the "refresh" button at the side.

After clicking refresh, scroll back up to the displays above. You should see a line near the middle of the page "Mouse (mm10) WholeTranscriptome qPCR Primers". Click on that to expand (or compress) the display of primers. You should see red and blue lines with arrows. Click on any of them to see details of the corresponding primer pair.

Unfortunately, the UCSC site only provides this function for human and mouse and only for genes that pass the testing described at the link above. Since Ins1 has only two exons, it does not pass the test (no primers from first exons are allowed, and only intron spanning primers are designed.)

You can still use the UCSC site to retrieve the sequences of the rat and mouse Ins1 genes, but you will have to use some other primer design software, such as primer Blast to design primers (or to test primers that you find by searching the published literature). Primer Blast is at:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/