miScript PCR Controls

miScript Primer Assays designed for a large panel of ncRNAs were evaluated to determine their amplification efficiencies and the variability of expression levels of the target ncRNAs in various cell and tissue types. Sequence conservation between human, mouse, rat, and dog ncRNAs was also studied to design miScript PCR Controls that can be used in all 4 species. miScript Primer Assays for 6 ncRNAs were selected as miScript PCR Controls. They fulfilled the following criteria:

• Endogenous reference ncRNA targets which show a relatively constant expression level across a variety of cells and tissue (see figure Constant expression levels of RNAs targeted by miScript PCR Controls)
• Amplification efficiencies close to 100% under standard conditions
• Equal performance in human, mouse, rat, and dog providing flexibility to use the same control for all 4 species

For accurate and reproducible results in real-time PCR analysis, it is necessary to normalize the amount of target miRNA by using a suitable endogenous reference RNA. This is known as relative quantification and is an important control which corrects for factors that could otherwise lead to inaccurate quantification. These factors include variation in quantity of input RNA, possible RNA degradation or presence of inhibitors in the RNA samples, and differences in sample handling. Normalization also allows results from different experiments and samples to be compared directly.

Features of a good control

An ideal endogenous reference RNA for normalization of data in real-time PCR quantification of miRNA should have the following features:

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.

ΔΔC_T method for relative quantification

The ΔΔC_T method is commonly used for relative quantification when working with the miScript PCR System. This comparative method relies on comparing the differences in C_T 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 (C_T) obtained with the miScript PCR Control is used to normalize the data as shown below.

Firstly, the threshold cycle (C_T) for each sample is determined. Next, the ΔC_T value is calculated. The ΔC_T value is the difference between the C_T value of the target miRNA and the CT value of the endogenous reference ncRNA:

ΔC_T = C_T (target miRNA) - C_T (endogenous reference ncRNA)

In the next steps, the ΔΔC_T value and the normalized target expression are calculated:

ΔΔC_T = ΔC_T (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 ∆∆C_T 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.

miRNA quantification and profiling