Another very useful in silico PCR tool is at the human genome browser gateway

http://genome.ucsc.edu/

Selecting  the PCR drop down menu will take you here:

http://genome.ucsc.edu/cgi-bin/hgPcr?command=start 

I often use the UCSC in silico PCR site in conjunction with the Primer 3 primer selection site:

http://bioinfo.ut.ee/primer3-0.4.0/primer3/ 

for primer design

I would be careful about selecting any primers from Primer depot or PrimerBank.  I've seen just as many bad designs as good designs there, and really what you want to develop is a method that works for you, rather than depending on what has been "traditionally" used (often a bad choice!)  If you do get primers there, you should subject them to all of the testing that you would do for preparing new designs.

And, of course, if your gene has no introns, then any RT-qPCR primer pair that you design will also amplify genomic DNA.  In those cases, you need to be sure that there is as little genomic DNA contamination in your RNA prep as possible.  You may need to treat your RNA with DNAse BEFORE you reverse transcribe it and there are a variety of commercial kits available for that purpose.

I should mention that I am not working with humans, but parasite. There's a database to obtain gene information (as well as NCBI) and I use IDT's PrimerQuest to design my primers. My question was regarding which sequence to use to design my primers since the database provides both mRNA and genomic sequence. Some times it doesn't matter which one I use since some genes I've come across do not have introns, but I came across one gene that did and was unsure as to which I should design my primers with.

In any case, thanks for the links, and David, thanks for clarifying that gDNA will be amplified if my gene had no introns. I always treat my samples with DNase during the extraction step.

On the UCSC in silico PCR web page, there is a drop down menu under "genome".  (Human is just one option of over 50.)  Sounds like your organism might not be there, but you could check.

It's too bad that all the other responders didn't read the question.  As I understand it, you are puzzled because, using primers that span an exon-exon junction, you are getting PCR amplicons from mRNA (expected) and genomic DNA (unexpected).  Now it's not exactly clear what you mean when you say primers spanning an exon-exon junction. Strictly speaking this means that the primer sequence itself spans an exon-exon junction in the predicted mRNA.  My answers that follow assume that this is indeed what you meant.

I would not expect your primers to align with the genomic sequence because only half of the primer (each side of the exon-exon junction) will align to its corresponding genomic position and the alignment parameters likely don't support alignment of such short sequences (otherwise you would get too many hits).  In this case, the other primer may have aligned to another locus.

One would not expect that using genomic DNA as a template and one primer spanning an exon-exon junction would allow amplification. Thus, it's possible that the 3' end of your primer managed to anneal at the corresponding position even though the conditions were thermodynamically unfavorable (believe me, it happens).  Alternatively, it's possible that you amplified a non-target locus of a very similar size (believe me, it happens).  You should sequence your product to be sure.

Finally, whether you use the mRNA sequence or gDNA to design primers, depends on your purpose and how much you know about your target locus.  If you do not know the gene structure (intron presence, etc.), you should use the mRNA to design primers for amplification when your original starting material was RNA and gDNA for when the original starting material was DNA.

In addition to all the advises given here, I'd suggest trying this software from Roche. it is really useful, I've used it and  got 99% specific amplification success of the genes I studied; I failed only one gene which is particularly complex (weakly expressed and has multiple alternative splices) ! 

http://lifescience.roche.com/shop/CategoryDisplay?catalogId=10001&tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1.

Also, if you are extracting RNA using the RNeasy® Micro Kit (or others), wich includes a DNase treatment step, my advise is to treat again your RNA with DNase before performing the RT, since some DNA molecules could bind the membrane of the column and therefore couldn't be degraded with the 1st DNase treatment. this step is important especially when you quantify intronless genes.

Another thing that you may do, is to perform a dissociation stage when you test your primers for the first time. this will give you an information on the specificity of your primers.

that's it!

Good luck

Thank you Mark. This was helpful.

Salah, how would I treat the RNA with DNase again? Do I add all my RNA through an Rneasy column (without centrifuging) and follow the same steps I followed for the treatment during the extraction step? This includes adding the DNase, incubating, and then adding the wash buffer, followed by the elution step. OR Do I add my RNA to the column, add the DNase, incubate, and spin?

Also, is the dissociation stage the same thing as a melt curve? If so, I do perform this.

No, you don't need to repeat that step. Instead, simply take 8 ul of your RNA solution (adjust according to your RNA concentration) + 1 ul DNase I + 1ul 10x DNase buffer (from Ambion or Fermentas). incubate 30min at 37C than inactivate the DNase by adding 1 ul EDTA (25 mM) and incubating 10min at 75C. finally proceed to RT in the same tube. it is suitable to use a PCR machine for all these steps (DNase treatment and RT), it is just more convenient!

Yes Dissociation stage = Melt curve.

Salah

Does it matter which DNase I use? I use Qiagen's, which is 10ul of Dnase + 70ul RDD (DNA digest) buffer per sample during the extraction step

It doesn't sound like DNA contamination is the problem in your case O because, from your question, it sounds like you are experiencing unexpected bands with the DNA template.  This being said, whenever you do Reverse transcriptase PCR, you should always run a no reverse transcriptase control to make sure that you don't have DNA carryover in your RNA preps.

It is depend of what you targeted - if you evaluating expression level you need to look at mature mRNA. In some cases this allow differentiation between mRNA and DNA target

It really depends on what is the objective of designing primers. In case you want to look at gene expression, or clone out the cDNA,  you would like to design primers within the exons. 

If you need to carry out gene expression study it is highly advised to design you primers with an intron spanning to avoid skewing your results with any possible DNA contamination. the reverse primers (if this is the primer that don't align) won't align if you align it as such, you need to reverse complement you sequence.

Unfortunately for the intronless gene you haven't another choice but designing the primers within the exon. make sure you treat your RNA with DNAse before carrying out your RT.

Here is a software that you could use for your design; it is really efficient!

https://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1

hope it will be helpful

If the primer you designed is spanning the junction, it is normal it doesnot align your genomic sequence but it should align the mRNA.

As for your second question, yes it is normal. Since you do not have intrones, the cDNA should have the same sequence than the gDNA, so that would be the excpected result