how to design the primers for Real-Time PCR?? - I can't find the proper ones... (Mar/06/2009 )

I have to design a set of primers for the isoforms of a certain gene in Arabidopsis in order to find their expression patterns.
These isoforms are highly homologous in the center of the cDNAs, so i can not look for primers in these homologous regions, right?

so i tried in the 5' end and the 3' end(i don't know if it is right to look for primers in the 3' UTR region of cDNAs, can you tell me ?).But there are soooooo many repeats of bases in these two regions that whatever primers i picked up had seemed to be unspecific when do BLAST with Arabidopsis genome(usua. there were 10 unspecific pairings )

the software i uesed:Primer3 online; Tair PCR;
the parameters i used : Tm:62-64 degree centigrade; product length:100-200bp; primer length:22-24bp;
GC%:30-60;

I really need help.....

the best software in the whole world is at NCBI....

it checks your sequence and compares it with all other sequences on NCBI to give you a correct set of primers which are specific for your gene or region of interest...it's called primer-BLAST....trust me it's the best:

http://www.ncbi.nlm.nih.gov/tools/primer-b...K_LOC=BlastHome

Curtis on Mar 6 2009, 09:30 PM said:

the best software in the whole world is at NCBI....

it checks your sequence and compares it with all other sequences on NCBI to give you a correct set of primers which are specific for your gene or region of interest...it's called primer-BLAST....trust me it's the best:

http://www.ncbi.nlm.nih.gov/tools/primer-b...K_LOC=BlastHome

Another Q:

The Tm parameters i set on the websit you suggested is 62-64 degree centigade, then when i do real-time PCR, should i follow it or use other formulations to work it out?

you can adjust the Tm on the online software to whatever you want....is that what you are asking?.....it's up to you how to set it. the software is giving you the recommended Tm.

hi, Curtis, thank you very much! i'd like to ask another 2 questions:

1.when doing real-time PCR, if you want to distiguish the pcr products of the mRNA from the pcr producsts of the genomic DNA contaminant, you'd better make sure that the pcr products are astride at least one intron, so that the length of the correct pcr product would be shorter than that of the pcr product of comtamimant DNA,right?

2.i' ve designed nearly all the primer pairs with the NIH-primer-blast as you suggested, but i failed to find out the proper one for one isoform from the 10 results that the NIH-primer-blast gave. What do you think shoud i do? maybe changing the searching parameters?

do you paste the cDNA sequence of your mRNA into the software?....this way you wouldn't need to worry about intron and exon.....I don't have problem with DNA results as the software only gives me primers for cDNA

do not change all parameters.....I usually change the amplicon size only to give me less than 100bp primers.

I assume that the homolog region in the middle is causing problem for you, right? that is why you get primers which can amplify all isoforms....why not try other regions?

for my case, it is not important if the primers amplify other isoforms or not. all are fine for me.

Have you checked if there are primers in any of the databases?
Like:
atrtprimer
RTPrimerDB
and so on...