You can try to use Prime 3 to design your primers, it worked for me (see the website: http://primer3.sourceforge.net/). I have also used the Primer3Plus (see website: http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/).

Dear Vishnute,

As I understand, You need to design custom primers for real time PCR. Do you consider TaqMan or only Sybr? I strongly recommend you to use TaqMan to estimate of gene expression (mRNA).

For me, first of al you should present for yourself exact structure of your gene with introns and exons. It is very comfortable for me to use VectorNTI, but if you can not an opportunity to use it, please use Your preferable soft.

If you haven't any soft - you can easily do it in powerpoint or even on the sheet of paper and intefrated functions of NCBI. But you should view the last 20 bp in each exon end and next exon start: ex1/ex2 connection with 40 bp (20 bp from the end of ex1 and 20 from the start of ex2).

Next, you have to choose your future TaqMan probe on the base of ex1/ex2 connection. If you use LNA or MGB (provided by companies that can produce your you primers after your design) you can use only 15 bp to do your probe. But if no (cheeper) you should use about 25-30 bp (depends on the sequence) to receive Tm about 65oC. To check probe Tm please use https://www.idtdna.com/pages/tools/oligoanalyzer of something by your choice.

After probe design (may be it is not a final probe because of subsequent analisys and quality estimation) you have to use primer3 like Mohammed said, and design forward and reverse primers in automatic way by soft.

Arter that your primers Tm (it is preferable) should be about 59-61oC and your probe Tm about 65-70oC.

Finally, using https://www.idtdna.com/pages/tools/oligoanalyzer you can estimate dG of your primer/primer primer/probe hetero and selfdimers. If dG0

To check potential reaction effectiveness your should use http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form for your primers and probe, amplicon. It provide you information about secondary structures under experimental conditions and after that you can correct conditions of reaction to increase effectiveness.

As your understand it is only general information. The best way to design for me. But of course you can easily design your primers with commertiol soft or with a help of appropriate companies, like Primetech (they provide full volume of work from design to reaction optimization) http://buyolig.com/

I hope that it will usefull for you.