QPCR analysis using qbase+

qbase+ introduction
2 Topics
Installation and Licence
Training Material
Experiment Design Exercises
7 Topics
Exercise 1: Simple Gene Expression Study
Exercise 2: A Large Study
Exercise 3: How to Fill Plates?
Exercise 4: A Growing Study
Exercise 5: A Diagnostic Copy Number Screen
Exercise 6: Fix Experiments with Bad or Missing Data
Exercise 7: Dilution Series for Calculating Amplification Efficiencies
Primer Design Exercises
5 Topics
Criteria for qPCR Primers
Fruit Fly Tap Gene: Designing qPCR Primers using PrimerBLAST
Fruit Fly Tap Gene: Analyzing primer characteristics using OligoAnalyzer
Human F9 Gene: Designing qPCR Primers using PrimerBLAST
Human F9 Gene: In Silico PCR in the UCSC Browser
Gene Expression Analysis
10 Topics
Loading Data
Adding Annotation to Data
Specifying the Aim of the Experiment
Checking the Quality of Technical Replicates and Controls
Considering Amplification Efficiencies
Normalization
Scaling
Visualizing the Results
Statistical Analysis
Run7
Analyzing Data from Different qPCR Experiments over Time
2 Topics
Creating a New Experiment
Analyzing the data
Selecting reference genes: exercises
8 Topics
About RefGenes
Starting the RefGenes Tool
The Genevestigator User Interface
Using the RefGenes Tool to Find Reference Genes
Finding Candidate Reference Genes in the Free Version of Genevestigator
Finding Candidate Reference Genes in the Commercial Version of Genevestigator
Exercise 1: Reference Genes for Mouse Liver
Exercise 2: Reference Genes for Human Heart
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Visualizing the Results

One of the things you can select to do on the Analysis page is viewing the relative expression levels (= scaled NRQs) of each of the genes in a bar chart per gene. It is recommended to visualize your results like this.

It is possible to view the relative expression levels of all genes of interest on the same bar chart. You can use this view to see if these genes show the same expression pattern but you cannot directly compare the heights of the different genes because each gene is independently rescaled!

How to visualize single gene expression bar charts?

Select Visually inspect results For individual targets on the Analysis page and click Finish.

How to visualize the expression levels of Palm in each sample?

Select Visually inspect results For individual targets on the Analysis page and click Finish.

The Target select box allows you to select the gene you want to view the expression levels of. Relative expression levels are shown for each sample. Error bars are shown and represent the technical variation in your experiment (variation generated by differences in amounts pipetted, efficiency of enzymes, purity of the samples…).

You see that Palm has a low expression level and a very large error bar in Sample05 because the two replicates of this sample had very different Cq values. You can group and colour the bars according to a property.

How to group the bars of Palm according to treatment (so treated at one side and untreated at the other side)?

In the Grouping section you can specify the property you want to group by.

How to view average expression levels in each group?

In the Grouping section you can choose to plot individual samples as shown above but you can also choose to plot group average expression levels.

The error bars that you see here represent biological variation and will be used later on in the statistical analysis. The error bars are 95% confidence intervals which means that they represent the range that will contain with 95% certainty the real average expression level in that group of samples. The nice characteristic of 95% confidence intervals is the following:

  • if they do not overlap you are sure that the expression levels in the two groups are significantly different, in other words the gene is differentially expressed
  • if they do overlap you cannot say that you are sure that the expression levels are the same. You simply don’t know if the gene is differentially expressed or not.

Assess the effect of switching the Y-axis to a logarithmic scale for Palm

In the Y axis section you can specify if you want a linear or logarithmic axis. As you can see you do not change the expression values, you just change the scale of the Y axis. Switching the Y-axis to a logarithmic scale can be helpful if you have large differences in NRQs between different samples.

Assess the effect of switching the Y-axis to a logarithmic scale for Flexible

Switch to the bar charts of Flexible. By switching the Y-axis to logarithmic you can now see more clearly the differences between samples with small NRQs.