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Primers for qPCR


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3 replies to this topic

#1 baab-try

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Posted 16 March 2011 - 10:59 PM

Hi all.

Very new to the field and just starting things.

I need to follow the gene expression in a plant using real time RT-PCR. Where do I really need to start? I.e, how will I design the primer for a plant whose sequence is not known and similar works have not been done on the plant (as far as I am aware)?

Pls advice, cos for now I am at a lost space! <_< thanks and appreciate any assistance.

#2 phage434

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Posted 17 March 2011 - 06:28 AM

You need to find the sequence of the gene of interest in the plant you are working with. One way of doing this is to find the gene in similar plant species, and identify highly conserved amino acid motifs. You can then design degenerate primers for these motifs, and amplify the gene from your plant. You sequence the gene, then develop more specific primers for qPCR for that gene.

Another approach is to do a library of fragments, then hybridize the extracted DNA (colony blot) with a similar gene or probe. Identify the clone, sequene, and then develop a specific primer pair.

None of these are easy or simple.

#3 baab-try

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Posted 17 March 2011 - 11:57 PM

Thanks a lot for that. I can see some light now. Plz again, I have 2 accessions of the same plant species. Does that means the same work for each of the accessions?

Thanks.

#4 Harvey

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Posted 18 March 2011 - 08:02 AM

Huh! Phage434 had a bright idea.

And in my opinion, you should figure out the specific gene for expression analysis. Then carry on your design work.

I think the first strategy is more feasible and time-efficient




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