Hello, I just started using Light Cycler 480 by Roche and I'm starting to get how it works. But one thing I can't understand is why do I get different Cp when i use "Basic Relative quantification" or "Advanced Relative quantification". Between the 2 configurations I don’t change anything. I keep E=2 as default.
I have one reference S16, and 3 target genes SMA, TGFb1, Collagen I a1 and a2. I also use a condition with no treatment for normalization (I put it as my internal calibrator).
Depending on what I use, my end results are different. For example in the basic quantification, my treatment seems to decrease TGFb1 but in the advance it's the reverse.
If someone is used to using LightCycler 480 software, could you help me understand better what's making my Cp change? So I can perhaps understand better what relative quantification I should be using.
Thank You for the help
In the Advanced analysis are you also selecting AbsQuant Fit Points instead of the default AbsQuant 2nd Derivative Max? The Basic analysis uses Fit Points methods to determine Cp, so you should keep that constant in both Basic and Advanced analyses. I think this should give you the same results then in the end. On pg 162 of their manual it says:
"LightCycler® 480 Software provides two methods for performing Absolute Quantification
analysis:
►► the Second Derivative Maximum method
►► the Fit Points method
Both methods use standard curves to calculate unknown sample concentrations, but each method determines a sample’s crossing point in a different way."
I am also just beginning to use this software, but what I understand is that it only makes sense to use the Advanced analysis if you have done serial dilutions and have an actual standard curve to give it for each gene so that it can calculate the REAL efficiency of each gene and apply that to calculating the Cp for each well. In my opinion, it is really important to know what your REAL gene efficiency is and to find this out you have to do a standard curve (for each gene). Only this way will your results make sense, because if you assume all your gene efficiencies equal 2 but they actually don't (or even if most do but one of your target genes is actually quite far from 2) then your analysis will be inaccurate.
SLIDE 20 in the link below shows the results of different gene efficiencies (efficiency of 2 means perfect doubling each cycle "100%")
http://slideplayer.com/slide/6893221/
I find it useful to look at the Demos that come with the software. For example, in the Demo Relquant Monocolor" file (from navigator view select root>roche>experiments). In the analysis tab they have already done one Basic Relquant Analysis and several variations of the Advanced Relquant Analysis, so you can see what their settings are in each and how this changes the results.
Lastly, I once called their phone number for technical help for the LC480 for questions about the software and it was really quick and helpful. Maybe try that too?
Good luck!
Jasmine
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