Please use this tool to see if you can find what we're looking for. http://www.srnaprimerdb.com/

Dear Mohammad,

I may not know the exact answer for your question, but I can help you with guidelines for primer designing. I have used these articles as a guide to design my primers and they were really good.

In Silico PCR Primer Designing and Validation: Anil Kumar and Nikita Chordia

10.1007/978-1-4939-2365-6_10

Optimization of Polymerase Chain Reactions: Haiying Grunenwald (Contains in depth details for PCR primer designs)

10.1385/1-59259-384-4:89

PCR Primer Design: David L. Hyndman and Masato Mitsuhashi

10.1385/1-59259-384-4:81

After referring the articles above I used Primer3 (https://primer3.ut.ee/) software to design primers.

I rechecked the primers using ensembl database https://asia.ensembl.org/index.html); to make sure the designed primers had only a single hit in the target genome.

To check further primer properties, I used this web tool (https://www.idtdna.com/pages/tools/oligoanalyzer). You can change Mg++ concentration and other parameters from the tool. Very good tool I may say.

I screened about 20 primers to filter and choose the best primer set.

As expected, they gave perfect bands when I did the PCR and gel electrophoresis.

Hope this info helps you. Good Luck!

Forward primer will be exactly same with the piRNA sequence (of course, select appropriately like any other primers, proper length, GC content etc.,).

If you are preparing the small cDNA by stem-loop method which is specific to your piRNA, then the reverse primer for qRTPCR will be specific to your stem-loop (for making stemloop primer check Saurabh's Saurabh Mandal link). On the other hand, if you are using cDNA synthesis kit with polyA tailing, reverse primers/Universal primer (whose sequence may be proprietary) are usually provided with the kit.

Stem-loop method is more specific compared to polyA tailing method. Hope this helps.