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High-Specificity miRNA QRT-PCR Detection - Details & Specifications

Agilent's High-Specificity miRNA QRT-PCR Detection kits provide qualified reagents for the polyadenylation of microRNAs (miRNAs) from total RNA and synthesis of first-strand cDNA from the tailed miRNAs (Figure 1) as well as a novel PCR enzyme formulation and QPCR detection reagents designed to give the utmost specificity for miRNA QPCR detection. The kit includes 2 modules consisting of: polyadenylation of the miRNA at the 3' end and reverse transcription to convert the polyadenylated miRNA to cDNA (miRNA 1st Strand cDNA Synthesis Kit) and cDNA detection by QPCR (High-Specificity miRNA QPCR Core Kit).

Figure 1: QRT-PCR Method to Detect miRNA Step 1: Addition of poly A tail to miRNA in total RNA. Step 2: Reverse transcription with an adapter primer (Oligo dT with unique sequence at the 5' end for universal reverse primer binding). Step 3: QPCR with miRNA-specific forward primer and universal reverse primer.

Powerful Sensitive Detection

Our High-Specificity miRNA QRT-PCR detection kit offers extreme specificity and sensitivity. Our kit can preferentially detect mature miRNA from as little as 15 ng of total RNA input on a variety of sample types. Our kit detects miRNA directly in lysed cells or in total RNA isolated from tissues, cell cultures, and FFPE tissues.

Highly Discriminative - Down to a Single Nucleotide

Our High-Specificity miRNA QRT-PCR detection kit discriminates between miRNAs that differ by as few as a single nucleotide as demonstrated by less than 1% cross reactivity between all human let-7 family members (Figure 2). To achieve this, we optimized our QPCR reagents for high specificity and developed a proprietary miRNA primer design approach.

Figure 2: High-Specificity miRNA QRT-PCR Detection Kit Assay sensitivity demonstrates single nucleotide discrimination. Specificity is comparable to or better than other commercially available kits. Our High-Specificity miRNA detection kit's specificity is higher than other commercially available kits. Relative detection (%) calculated based on CT difference between perfectly matched and mismatched assays.

Better Sensitivity and Specificity

Our High-Specificity miRNA QRT-PCR Detection kits have been optimized for the detection and quantification of miRNA from 15-250 ng of total RNA using EvaGreen® detection reagent that gives better sensitivity and specificity. EvaGreen is a superior fluorescent DNA stain for quantitative PCR (QPCR). Upon binding to DNA, the fluorescence of EvaGreen is several fold higher than that of SYBR® Green I while the dye shows very little inhibition to the PCR process (Figure 3). Unlike SYBR Green, which has been reported to be unstable, EvaGreen is highly robust. In addition, the absorption and emission spectra of EvaGreen are similar to that of SYBR Green I, which means that the same optical setting for SYBR Green I can also be used for EvaGreen.

Figure 3: EvaGreen® is highly stable. A solution of EvaGreen dye or SYBR® Green I dye at 1.2 µM in pH 9 tris buffer was incubated at 99°C over a period of 3 hours. The absorption spectrum of each solution is shown. Rox was added as a stable reference. (Provided by Biotium Inc).

For Research Use Only. Not for use in diagnostic procedures