Hi Mohammad,

first of all I think it is a good idea to verify the primer sequences before you use them because in my experience you have wrong primer sequences in papers once in a while.

What I did usually with primer sequences to verify them is first to do a Blast search. Make sure to choose the right data set (RNA or genomic) and to enter the organism.

As primer sequences are short you will usually get several hits, however if you blast both sequences (for the forward and reverse primers) you should get a hit in the same gene and/or genomic region. You should then verify if the amplicon size is correct.

Additionally, you can download the target sequence and use a program for DNA analysis to search for the primer sequence within the target sequence and/or align the primer sequence (I use ApE (A plasmid Editor) which is a free software and usually works fine for me).

However, this is getting more difficult is you look for what you call "methylated primers" - I assume you mean primers which are targeted against a DNA sequence after bisulfite conversion (please correct me if I am wrong). This means that in the primer sequence you have a T instead of a C for the fw primer and an A instead G for the reverse primer. You can still try to blast the sequence – however, this might not work due to mismatches. Nevertheless, you can still align these primers against the target sequence as some mismatches are usually tolerated here. Check if the sequence you find is fitting to the sequence for bisulfite converted DNA.

I hope this helps

Thank you for your response.

We got the reference sequence of our target gene from NCBI, and then used the Eukaryotic Promoter Database (EPD) to accurately identify and extract the promoter region.

To validate the primers we are planning to use (both those obtained from published studies and those designed using Meth Primer) we attempted to run them through NCBI’s Primer-BLAST. However, in many cases, especially for methylation-specific primers, the tool either fails to align the sequences correctly or returns no results at all.

regards

Reza

Hi Mohammad ,

sorry that it took me some time to come back to. I still think you still have difficulties with the mismatches in primers designed against a bisulfitte converted DNA Ssequence (methylated primer) and a more "manual" approach with a programm like ApE might work better. Additionally, it is also sometimes hard to find the right promoter region. Are you willing to share some of your primer data? Than I can try if it works better with ApE - however I can not promise that it will work.

KR Dörthe

Hi Dörthe,

Thank you very much for your kind reply, and I apologize for my delayed response.

You're absolutely right, designing primers for bisulfite converted sequences can be quite tricky due to the mismatches involved. I will definitely try to check the primers using ApE as you suggested and see if it helps improve the validation process.

If I face any difficulties or can't get it to work properly, I’ll share the primer data with you. I’d truly appreciate your help and feedback if you have the chance to take a look.

Thanks again for your support!

Best regards, Reza

Hi Mohammad,

if you have difficulties with the sequences you can share the primer data with me and I am happy to help.

KR Dörthe

Hi Dörthe,

Thank you very much for your time and kind support.

Best regards,

Reza