Trying to measure small levels of mRNA - (Mar/03/2010 )
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?
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.openbiosystems.com/Query/?q=BC018425 its 87.- EUR. This is a mouse gene.
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.
Blackeyed on Mar 4 2010, 10:09 AM said:
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.