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To obtain reliable miRNA expression data it is important that an experiment is designed to accurately subtract background signal, allow for appropriate normalization and assess inter-well variability. Every experiment performed with the FirePlex® miRNA assay includes:
Each custom or focus panel includes a positive control ("X-Control") and a blank ("Blank") by default. Endogenous controls may also be included.
Positive control particles bear probes for an miRNA-like target, X-Control, that is present in FirePlex Hybe Buffer. This control gives confidence that the assay was successfully implemented in every well.
Blank particles bear no probe, giving a baseline level of the background fluorescence in every well.
Endogenous controls are small nucleolar RNAs or miRNAs expressed at consistent levels under a variety of conditions. These controls are important for signal normalization between sample types and treatments. Users are given the freedom to select endogenous controls most appropriate for their applications and may prefer to include more than one endogenous control in their panel.
Our FirePlex® Analysis Workbench analysis software is designed to review the expression of each target in each well of your experiments and identify the best potential normalization targets from the available options. If, however, none of the targets included in your panel are detected in every sample at a sufficiently high level, none of the targets may meet selection criteria.
As a result, if your confidence in your choice of normalization targets is low, we suggest including at least three potential normalization targets in your custom panels; all focus panels include at least three potential normalization targets. Selection of targets that you do NOT expect to change is just as critical as selecting targets that you do expect to change.
Below are normalization targets that have worked well in our experiments, but your experience may vary:
|Circulating Assay potential normalizers|
|Human plasma/sera||let-7d-5p, let-7g-5p, let-7i-5p, miR-29b-5p|
snoRNAs are unreliable controls in plasma or serum, so miRNAs are recommended.
In order to obtain an accurate measurement of the background signal for each miRNA in a panel, it is necessary to run negative control wells where a carrier buffer is used in place of a biological sample. Furthermore, the use of multiple negative control wells allows users to estimate inter-well variability, giving more confidence in the results obtained. We strongly recommend the use of at least three negative controls per panel every time an assay is performed.
If you are running the FirePlex miRNA (biofluid) assay for the first time you may want to run negative controls both for the digest (by adding water to the lysis buffer) and for the assay as a whole (by adding water to the Hybe buffer in the first hybridization step).
The use of replicates gives statistical meaning to results by, for example, enabling the calculation of mean and standard deviation. Replicates can be performed at the stage of sample preparation (biological) or assay (technical).
Biological replicates are those in which the same conditions are used to treat and prepare samples from different sources. These replicates are important in determining the biological variation within a population. An example of this type of replicate is serum samples derived from three different mice injected with the same TNF inhibitor.
Technical replicates are those in which samples derived from the same source are assayed multiple times. These replicates are important in determining the reproducibility of the assay. Ideally, every assay has technical replicates.
FirePlex® is the new name for our Firefly assays.
FIREPLEX is a registered trademark in the United States and is an unregistered trademark elsewhere