Analyzing data from a geNorm pilot experiment in qbase+

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[ Main_Page | Exercises on using qbase+ ]

In a geNorm pilot experiment you analyze a set of candidate reference genes in a representative set of samples that you want to test in your final experiment. Based on the M-values and CVs that are calculated by qbase+, you can choose the genes that most satisfy the criteria for a good reference gene.

Exercise 1: reference genes for mouse liver

We come back on the 8 candidate reference genes that we selected for mouse liver:

  • 4 commonly used reference genes: ACTB, TUBB4B, GAPDH and HPRT
  • 4 candidate reference genes with very stable medium expression levels selected based on expression data coming from more than 600 microarrays of mouse liver samples using Genevestigator: Gm16845, MUSK, OTOP3, EDN3

We have measured their expression in a represetative set of 16 of our mouse liver samples, each in triplicate. We will now analyze the stability of these candidate reference genes in our samples.

Creating a new Experiment

Loading the data into qbase+

The data is stored in the RefGenes folder. It consists of 8 Excel files, one file for each candidate reference gene.
If you are not working on a BITS laptop, download and unzip the folder.

This file contains the data of the geNorm pilot experiment.
In the pilot experiment, 8 candidate reference genes were measured in 16 representative mouse liver samples.


SampleMouse.png

Analyzing the geNorm pilot data

It is of course better to include a dilution series for each gene to have an idea of the amplification efficiencies of each primer pair.

Click Finish.

These are artificial data. But when you read the paper by Hruz et al., 2011 you see that the genes that are selected by Genevestigator are often outperforming the commonly used reference genes.

Exercise 2: reference genes for human heart

Creating a new Experiment

Loading the data into qbase+

This file contains the data of the geNorm pilot experiment.
In the pilot experiment, 10 candidate reference genes were measured in 20 representative samples.


SampleHuman.png

Analyzing the geNorm pilot data

It is of course better to include a dilution series for each gene to have an idea of the amplification efficiencies of each primer pair.

Click Finish.



References: