For normalization of real-time PCR results in miRNA quantification in human, mouse, rat, and dog

  • Effective in human, mouse, rat, and dog
  • High amplification efficiencies of close to 100%
  • Constant expression levels in a variety of cells and tissues
  • Easy to order via GeneGlobe
For accurate and reproducible results in miRNA quantification by real-time PCR, it is necessary to normalize the amount of target miRNA by using a suitable endogenous reference RNA. This is known as relative quantification. miScript PCR Controls are primers that enable normalization of real-time PCR results in miRNA quantification studies using the miScript PCR System. miScript PCR Controls are available for a panel of 5 snoRNAs (SNORD61, SNORD68, SNORD72, SNORD95, and SNORD96A) and the snRNA RNU6B (RNU6-2). These controls are designed taking into consideration sequence homologies in human, mouse, rat, and dog so that the same control can be used for all 4 species. They can be ordered individually at the GeneGlobe Web portal.
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miScript PCR Controls are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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miScript miRNA PCR Array layout for 96-well plates.|Constant expression levels of RNAs targeted by miScript PCR Controls.|
Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells H1 and H2 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells H9 and H10 contain replicate reverse transcription controls (miRTC). Wells H11 and H12 contain replicate positive PCR controls (PPC). These controls are also included in 384-well plate and Rotor-Disc 100 format miScript miRNA PCR Arrays.|Total RNA from various [A] human, [B] mouse, [C] rat, and [D] dog tissues was used for expression analysis of snoRNA/snRNA using 6 miScript PCR Controls. CT values show that the expression level of each RNA was very similar in different tissue types.|
Performance

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
Principle

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:

A constant expression level across all samples in the study
A similar small size as the miRNA under study
A similar expression level in the sample as the miRNA under study
Should not be regulated under the experimental conditions
Primers for the endogenous reference RNA and the miRNA should have similar amplification efficiencies (close to 100%)

miScript PCR Controls meet the above criteria, enabling the ΔΔCT method of relative quantification of miRNA levels in human, mouse, rat, and dog using the miScript PCR System.

miScript PCR Controls target noncoding RNAs

QIAGEN provides a range of miScript Primer Assays targeting ncRNAs such as snoRNAs and snRNAs. Scientists at QIAGEN have systematically evaluated a wide panel of these miScript Primer Assays for various ncRNAs and selected 6 miScript PCR Controls for 5 snoRNAs and an snRNA (see table below). These controls are also found on every miScript miRNA PCR Array (see figure miScript miRNA PCR Array layout for 96-well plates).

Table. miScript PCR Controls
miScript PCR Control name* ncRNA official symbol ncRNA alternative name ncRNA type Expressed in human, mouse, rat, and dog
Hs_SNORD61_11 miScript Primer Assay SNORD61 snoRNA Yes
Hs_SNORD68_11 miScript Primer Assay SNORD68 snoRNA Yes
Hs_SNORD72_11 miScript Primer Assay SNORD72 snoRNA Yes
Hs_SNORD95_11 miScript Primer Assay SNORD95 snoRNA Yes
Hs_SNORD96A_11 miScript Primer Assay SNORD96A snoRNA Yes
Hs_RNU6-2_1 miScript Primer Assay RNU6-2 RNU6B snRNA Yes
* As a result of restructuring the web databases for GeneGlobe products, some assay names for miScript Primer Assays targeting snoRNAs and snRNAs have changed. The unique GeneGlobe catalog numbers for these products (i.e., MSXXXXXXXX) have not changed.

Important Note: The miScript II RT Kit contains 2 buffers: miScript HiFlex Buffer and miScript HiSpec Buffer. The following, previously available miScript PCR Controls cannot be used with cDNA prepared using miScript HiSpec Buffer:
Hs_RNU1A_1 miScript Primer Assay
Hs_RNU5A_1 miScript Primer Assay
Hs_SNORD25_1 miScript Primer Assay
Hs_SCARNA17_1 miScript Primer Assay
Hs_SNORA73A_1 miScript Primer Assay
If using these miScript PCR Controls, ensure that cDNA is prepared using miScript HiFlex Buffer.

For cDNA prepared using miScript HiSpec Buffer or miScript HiFlex Buffer, we recommend use of any of the updated miScript PCR Controls listed in the table above.

Procedure
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.

Applications

miRNA quantification and profiling

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