Dear all,
I am using REST software to analyse my qPCR data (based on Pfaffl method), let say I am checking for gene expression of Gene A and Gene B in two groups (the treatment group and the control group), the relative expression ratio of Gene A is 0.96 and Gene B is 1.77, what do these numbers represent? And by just looking at the relative expression ratio, how do I know if the gene is up- or down- regulated a lot or a little?
Thanks in advance.
JM
Isn't this stated in the REST manual? Or in the respective publication? There should be all formulas and all explanations. It seems superfluous to repeat that here. Or do you have a specific problem we can help to clarify?
Hi Dr. Wilhelm, thanks for the reply. I have actually gone through the user guidelines provided by Qiagen and the REST tool paper published in 2002, I understand that the expression ratio presented is relative to 1.0. As I am testing my data with the other tool (qBase), I got a negative value for my down-regulated gene (while REST gave me a value
If you read the user guides, it should actually be clear. I assume (! - I neither use these software nor have I read their manuals!) that qBase gives log fold-changes and REST given fold-changes. If so, then this is a simple transformation and it will not change the interpretation if you use one or the other. For reasons of a more straight-forward interpretation of the biological relevance and visualization I'd prefer log fold-changes. But: I further assume that both software have some more differences. As far as I know does REST try to adjust for gene-wise differences in amplification efficiencies. I am not aware if qBase does the same. However, if the algorithms are different, you must decide which algorithm will suit your problem and your data best. At the end you can still decide if you will present the results as fold-changes or log fold-changes.
Agreed with Jochen
I've used REST before, and it's quite straightforward. If you get relative expression < 1.0, that means there is negative fold changes.
I've not used qBASE, so no comment on that.
You need to pick the software that best fits your need and experimental setup. REST allows you to use multiple reference genes to get the average reaction efficiency, not sure if qBASE uses a different method to calculate reaction efficiency for reference genes.
@Jun, just to clarify:there is nothing like "negative fold changes". Fold-changes (FC) are by definition >= 0. Only log fold-changes (LFC) can be negative.(I am sure you meant it right, but you said it wrong)
You get LFC
@Jochen: Yes, that was a mistake on my part. What I meant was down-regulation, i.e. decreased expression. Values < 1 in REST indicate relative decreased expression, while values > 1 indicates the opposite.
Thanks for the correction.
You're wellcome :)
I did not want to nit-pick or something. I just really find it important to reduce potentially harmful confusion.
Thanks all for the reply. I've got my problem resolved already =)
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