5 replies to this topic

[spok]

I'm need to perform real-time PCR from paraffin tissue.  I understand that specific primers may be better for the cDNA synthesis.  Does anyone know of any programs that will help me pick a primer.  I prefer a free, online application if anyone know of any.  Thanks

[littlecell]

just visit http://perlprimer.sourceforge.net/ and download perlprimer and select real-time PCR subtype. it works well. you can download the DNA sequence of your interested gene with ensemble menu directly and design the extrone-spaned primer for real-time pcr. good luck.

[spok]

Thanks.  Great program.

[spok]

To design a gene specific primer for RT, is the primer to the 5 prime of the real time amplicon?

[pcrman]

spok, on Mar 14 2005, 01:22 PM, said:

To design a gene specific primer for RT, is the primer to the 5 prime of the real time amplicon?

If I am not wrong, the gene-specific primer for first strand cDNA synthesis has to be located 3' of your PCR amplicon and antisense to the coding strand, similiar to oligo-dT primer.

[spok]

Do you know if I can use the same reverse primer for both the RT and real time PCR and still get quantitative results?

If I design separate primers for the RT, can you suggest some conditions to specify in software?  Thanks