Analyzing the expression of miRNAs in qbase+

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microRNAs (miRNAs) are small non-coding (not translated into proteins) RNAs that regulate gene expression. They bind to complementary sequences in mRNA. As a result, these mRNAs can no longer be translated into proteins and miRNA-mRNA complexes are targeted for breakdown.

miRNAs are produced by transcription, so their production can be measured by qPCR and analyzed in qbase+ in the same way as the production of mRNA is.

The experiment consists of a single run: Run12
The expression of the following miRNAs was measured:

• 2 reference targets: miRNAs RNU24 and RNU48
• 2 targets of interest: miRNAs mir-92 and mir-628

There are two technical replicates per reaction.

Sample annotation can be found in a separate sample properties file.

Creating a new experiment

Create a new Experiment called miRNAProfiling in Project1
You can find the details on how to create a new experiment in Creating a project and an experiment

Loading the data

Import Run12.
The file is in 7900 AQ format, which is automatically recognized by qbase+. You can find the details on how to import the data file in Loading data into qbase+

To analyze and visualize the analysis results in a later stage, we are going to add a custom sample property: MYCN status.

MYCN is a transcription factor. Amplification and overexpression of MYCN is known to lead to tumorigenesis. The experiment that we are analyzing tries to assess whether the expression of the two target miRNAs of interest is related to the copy number of MYCN.

Add sample annotation from the sample properties file.
You can find the details on how to add a custom property from a sample properties file in Creating a sample properties file

Analyzing the data

Choose the type of analysis you want to perform.
We're still doing a gene expression analysis: instead of looking at the expression of genes, we are assessing the expression of miRNAs but the principle is the same.

Check controls and replicates.
Take the default minimum requirements for controls and replicates. You see that all replicates meet these requirements. Controls were not included in this analysis.

We have done previous qPCR experiments with these primer pairs and we known from these previous experiments that

• amplification efficiencies for the reference targets are 95%
• amplification efficiencies for the targets of interest are 98%

Use these settings for the amplification efficiencies.
First specify that you want to use assay specific amplification efficiencies. Then define the target specific amplification efficiencies: Select to Use custom efficiencies Type the efficiencies in the provided efficiencies table. Remember an efficiency of 95% is expressed as 1,95 in qbase+.

Appoint the reference targets.
RNU24 and RNU48 are the reference targets. You can find the details on how to appoint reference targets in the Normalization section of Analyzing gene expression data in qbase+

Is the stability of the reference targets ok ?

In the Reference target stability window the M and CV values of the reference targets are shown in red so the stability of the reference targets is not ok. However, we only have two reference targets so it is impossible to leave one out. This again shows the importance of using a minimum of three reference targets. You can find the details on how to check reference target stability in the Normalization section of Analyzing gene expression data in qbase+.

Which scaling strategy are you going to use ?
Since you have a custom property, MYCN status, dividing the samples into two groups: single copy and amplified, it seems logical to use the average of the single copy group for scaling. You can find the details on how to specify the scaling strategy in the Scaling section of Analyzing gene expression data in qbase+

Look at the target bar charts.

In the target bar charts group the samples according to MYCN status.
You can find the details on how to group the samples in the Visualization of the results section of Analyzing gene expression data in qbase+

Is there a statistical significant difference of expression of the targets of interest between the two groups ?

You only have 6 replicates per group so you cannot test if the data comes from a normal distribution. Qbase+ will assume they're not normally distributed and perform a non-parametric Mann-Whitney test. The p-value of both miRNas is smaller than 0.05 (red) so it has a statistically significant difference in expression levels in amplified MYCN samples compared to single copy MYCN samples. When you look at the mean expression levels (green) you see that miR-628 has a significant lower expression level in amplified MYCN samples whereas miR-92 has a significant higher expression level in amplified MYCN samples You can find the details on how to compare the means of the two groups in the Statistical analysis section of Analyzing gene expression data in qbase+.

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