How to choose miScript PCR Controls
Choosing an appropriate miScript PCR Control for an miRNA quantification experiment is similar to choosing an appropriate housekeeping gene for normalization when quantifying mRNA. For each sample type or treatment under study, it is necessary to verify that the miScript PCR Control target ncRNA is not regulated under the experimental conditions. In addition, a miScript PCR Control for an ncRNA that shows a constant level of expression and similar abundance to the target miRNA should be chosen. miScript PCR Controls can be ordered individually at the GeneGlobe Web portal. In addition, they are provided on all miScript miRNA PCR Arrays and miScript miRNA QC PCR Arrays.
ΔΔCT method for relative quantification
The ΔΔCT method is commonly used for relative quantification when working with the miScript PCR System. This comparative method relies on comparing the differences in CT values obtained with normal versus experimental samples. Real-time PCR is performed using a primer (miScript Primer Assay) for the miRNA of interest side by side with reactions using a miScript PCR Control, which detects a snoRNA or snRNA. The threshold cycle (CT) obtained with the miScript PCR Control is used to normalize the data as shown below.
Firstly, the threshold cycle (CT) for each sample is determined. Next, the ΔCT value is calculated. The ΔCT value is the difference between the CT value of the target miRNA and the CT value of the endogenous reference ncRNA:
ΔCT = CT (target miRNA) - CT (endogenous reference ncRNA)
In the next steps, the ΔΔCT value and the normalized target expression are calculated:
ΔΔCT = ΔCT (sample [e.g., disease cells]) - ΔCT (calibrator [e.g., normal cells])
Normalized target miRNA expression in sample = 2-ΔΔCT
Finally, the normalized level of expression in the calibrator is set to be 100%:
% change in expression = (Normalized target miRNA expression in sample x 100)% - 100%
miScript miRNA PCR Array data analysis
The miScript miRNA PCR Array data analysis tool automically performs quantification using the the ∆∆CT method of relative quantification as well as interpretation of the control assays. Results are presented in a tabular format, a scatter plot, a three-dimensional profile, and a volcano plot (when replicates are included). The complimentary analysis tool is individually tailored for each miScript miRNA PCR Array and can be used in a Web-based or Excel format.