Hi migmar

if you want to do your PCR without any trouble, please dont use designed primers..

get your correct RBM3 gene sequence from NCBI, use NCBI-PRIMER BLAST to obtain some strong suggestion and check your primer in GENERUNNER..

be succeed

I compltely concur with Amin

• I never trust primers from papers as many do not work as the author claim; especially for a particualr gene but in a different treatment/tissue context
• Moreover, if the papers are more than 5 tears old transcript models change and so you need to access Contemporaneous Ref Gene Models to design primers
• Also, cross Ref transcript models in NCBI GenBank with Ensembl and only design primers to transcripts that are present in both
• Finally, apart from the usual checks in relation to primers like screening for primer dimers and hair pin loop remember to BLAST search primers for specificity
• To that end find attached a detailed dprotocol to how you can design optimal primers using free ware tools like IDT
• In addition, I have provided a detailed protocol as how to locate Ref gene transcrritps and then design primers using NCBI primer BLA: This tool is particualrly effective when you wish to design primers for specific isoforms
• To capture all nascent transcripts in your systemn find it is easier to identify gene transcript using NCBI and Ensembl and then import those transcripts into tools from IDT that will specifically make primer pairs to your transcript and map them to your transcript sequence automatically

I fully agree with the previous answers: design the primer by your own!

One more remark: If your really want to use published primer sequences, do a BLAST and check the parameters! This will quickly give you a hint whether it is worth trying it or not.

@Laurence Stuart Darwins-Hall

I had blast the primers in NCBI database and 100% query was shown and even performed oligoanalyser using IDT and everything came well till then.

I had gone through different papers and one paper used same gene using same primers but had set different time for PCR i.e. denaturing, annealing, and elongation which is very less compare to the time interval that i have set.

I would like to know whether that makes difference avoiding the primer dimer formation.

Your suggestions will be highly acknowledged.

I will answer this question monday GMT :-)

Dear Migmar

Primer dimer formation i not particualrly affected by PCR conditions

I do generlly include 5% DMSO in y sybr green reaction which can help

I would also use a dentauring temp of 96C not 95C

However, he best way to try and eliminate primer dimers is optimal primer formation

Hope that helps

Laurence

@Laurence Stuart Dawkins-Hall

I would like to bring in your notice that the paper had used random hexamers for cDNA conversion while i have used oligodTs for the cDNA conversion. Can that be a reason for observing a primer dimer in the qPCR that i have conducted? Your suggestions are highly valuable to me.