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designing gene-specific primer for cDNA synthesis


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#1 spok

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Posted 14 March 2005 - 02:33 AM

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

#2 littlecell

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Posted 14 March 2005 - 05:59 AM

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.

#3 spok

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Posted 14 March 2005 - 11:18 AM

Thanks. Great program.

#4 spok

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Posted 14 March 2005 - 11:22 AM

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

#5 pcrman

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Posted 14 March 2005 - 12:29 PM

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.

#6 spok

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Posted 15 March 2005 - 05:35 AM

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




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