In order to carry out accurate assays of miRNA and gather reliable expression data, the experiment must be set up correctly so that the signal can be normalized and the background signal can be subtracted. The variability between wells should also be assessed.
This is made simple using the FirePlex® miRNA assay. Every experiment using the FirePlex® assay includes replicates and controls (both negative and within each well)
Using replicates enables the determination of the mean and standard deviation of the results; this provides statistical weight to the data. Replicates can be used during any part of the assay (technical) or the sample preparation procedure (biological).
Technical replicates involve assaying samples from the same source multiple times; this is done to see how reproducible the results are. For good data, researchers should always run technical replicates.
Biological replicates involve assaying samples from different sources under the same conditions and prepared using the same method. This is done to provide insight into the biological variation within a population.
An example of a biological replicate is serum samples taken from three individual mice that have been administered the same TNF inhibitor.
Controls Within Each Well
Every Abcam panel provided contains a blank, labeled “Blank”, and a positive control, labeled “X-Control”. In addition, endogenous controls can also be provided.
The blank control contains nothing, which allows it to provide a baseline level that shows the background fluorescence of the well itself.
Positive controls contain an miRNA-like target probe, called X-Control, which is also present in the FirePlex Hybe Buffer. Using X-Control allows researchers to know that assays have been carried out correctly.
Endogenous controls are needed to normalize signals between different sample treatments and sample types. The controls consist of small RNA fragments or miRNAs which are expressed consistently regardless of the conditions.
Researchers select which endogenous controls are needed for their research and in some instances may want more than one.
Abcam’s provided analysis software, FirePlex® Analysis Workbench, can review target expression for each experiment well and, using this data, will determine the best normalization targets. However, if no targets in the experiment are detected at a high enough level, none of the targets will be selected.
Due to this, if you have low confidence in the normalization targets chosen it is good practice to include three or more potential normalization targets in your custom panel. As a standard, all focus panels include at least three potential normalization targets. To have useful results, having targets which are not expected to change is just as important as having targets that are expected to change.
Please find below examples of normalization targets that have worked in Abcam experiments:
Circulating Assay Potential Normalizers
Human plasma/sera miR-29b-5p, let-7d-5p, let-7g-5p, let-7i-5p, miR-29b-5p
miRNAs are recommended as snoRNAs are not reliable controls in plasma or serum
Negative Control Wells
To make an accurate background signal measurement for each miRNA sample a negative control well measurement must be made, which uses just carrier buffer with no biological sample. In addition to this, running multiple negatives accounts for variations between wells, which provides results with more confidence. For this reason, Abcam recommends at least three negative controls are used per assay.
If this is the first time you have ran a FirePlex miRNA with plasma or biofluids, you might want to run negative controls for the assay as a whole (in the first hybridization, by the addition of water to the Hybe buffer) and for the digest (by the addition of water the lysis buffer).
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