Comments on question 1

Although you may have validated your reference genes before it is
still advisible to verify the stability of these reference genes in
the actual experiment (= quality control).
It is normal that the NF vary from sample to sample. This is the
reason why you need to perform normalization. A 60-fold difference is
quite large. Have you normalized your RNA before proceeding to the RT
step?

Comments on question 2

You can get a quick indication of the reliability of your assay by
looking at the reproducability. Your technical replicates should be
very similar (delta Ct < 0.5 cycle). A very thorough check to
evaluate wether your assay still performs as it should, you can create
a dilution series and see wether you still obtain a (log-) linear
relationship between your Ct values and the input quantities at these
higher cycle numbers.

--- In qBase@yahoogroups.com, "isabelle.schrauwen"
<isabelle.schrauwen@...> wrote:
>
> Hi,
>
> I have 2 questions:
>
> 1. I have samples with different quality I want to compare. I used 2
> ref genes that are normally very stable in this tissue (we tested this
> before), after calculation of the NF, it ranged from 0.1 to 6 between
> the samples, illustrating clearly that there is a difference in
> quality and/or quantity. Will it matter to use more reference genes
> and is there even a reliable method to compare samples with a
> different quality and/or quantity? These are the only samples I have
> and it is difficult to collect new ones.
>
> 2. I have a gene I want to investigate that has a very low expression
> in the tissue I have RNA from. In all samples the Cp value is around
> 34-38 (in non-diluted cDNA)? Is it still reliable to test expression-
> differences? What is the maximum Cp value for a reliable analysis.
>
> Kind regards
>