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Trying to measure small levels of mRNA


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#1 Blackeyed

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Posted 03 March 2010 - 09:03 AM

Hey,

I'm trying to measure the levels of Wnt5a mRNA in lung tissue. I've designed primers and i'm trying to determine my efficiency but I only measure mRNA is my samples if it is a 1/5 or 1/10 dilutions.
If I make more dilutions the mRNA is not anymore detected. So how do I determine the PCR efficiency and
determine that my primers are working?

#2 tea-test

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Posted 03 March 2010 - 09:31 AM

You can do a dilution curve with purified pcr product or, if available, with plasmid. If you don't have the gene in a plasmid you may be able to order it, cDNA clones are often not very expensive.

Edit: I did a quick research and I found a full length clone http://www.openbiosy...ery/?q=BC018425 its 87.- EUR. This is a mouse gene.

Edited by tea-test, 03 March 2010 - 09:38 AM.

tea-test: The artist formerly known as Ned Land

#3 Blackeyed

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Posted 04 March 2010 - 01:09 AM

I don't know if ordering a cDNA clone is possible. We are working with sheep-material and
the sequence of the gene is not yet known. So I designed my primers on conserved regions of
other animals hoping that it's going to work in sheep.

#4 tea-test

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Posted 04 March 2010 - 03:09 AM

I don't know if ordering a cDNA clone is possible. We are working with sheep-material and
the sequence of the gene is not yet known. So I designed my primers on conserved regions of
other animals hoping that it's going to work in sheep.



then you can use purified pcr product directly or cloned into a vector.
tea-test: The artist formerly known as Ned Land




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