2 replies to this topic

[pcrmossad]

Hi all,

I am using RT-PCR for validating microarray data for some time now. usually I use 1, 0.1, and 0.01 dilutions to generate my standard curve. in rare cases I have used 0.2 .  

my question is should I always use these dilutions? cant I use 1, 0.5, 0.25 etc dilutions to generate a standard curve? what are the disadvantages of using this kind of dilutions apart from wasting control samples?

thanks in advance.

[Trof]

You should always have standards that span the area where your samples are. So if you except samples to be maximum 10 fold downregulated, you don't need to do 0.01 dilution in your standard curve.
Usualy more wider dilutions are used because they span all possible concentrations, but if diferent dilutions have different amplification efficiency (due to inhibition for example) it may skew the slope of the standard curve. In that case for concentrations around 0.5 - 0.25 the standard curve with 0.5 and 0.25 dilutions could be more accurate. It really depends on the range of your samples.

[pcrmossad]

Trof, on Apr 20 2010, 02:39 PM, said:

You should always have standards that span the area where your samples are. So if you except samples to be maximum 10 fold downregulated, you don't need to do 0.01 dilution in your standard curve.
Usualy more wider dilutions are used because they span all possible concentrations, but if diferent dilutions have different amplification efficiency (due to inhibition for example) it may skew the slope of the standard curve. In that case for concentrations around 0.5 - 0.25 the standard curve with 0.5 and 0.25 dilutions could be more accurate. It really depends on the range of your samples.

thanks again !