Real-time quantification of microRNAs by stem-loop RT-PCR

Abstract

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.

Figure 1

Schematic description of TaqMan miRNA assays, TaqMan-based real-time quantification of miRNAs includes two steps, stem–loop RT and real-time PCR. Stem–loop RT primers bind to at the 3′ portion of miRNA molecules and are reverse transcribed with reverse transcriptase. Then, the RT product is quantified using conventional TaqMan PCR that includes miRNA-specific forward primer, reverse primer and a dye-labeled TaqMan probes. The purpose of tailed forward primer at 5′ is to increase its melting temperature (Tm) depending on the sequence composition of miRNA molecules.

Figure 2

Dynamic range and sensitivity of the TaqMan lin-4 miRNA assay. (A) Amplification plot of synthetic lin-4 miRNA over seven orders of magnitude. Synthetic RNA input ranged from 1.3 × 10⁻³ fM (equivalent to 7 copies per reaction) to 1.3 × 10⁴ fM (7 × 10⁷ copies per reaction) in PCR; (B) Standard curve of the lin-4 miRNA.

Figure 3

Correlation of total RNA input to the threshold of cycle (C_T) values for eight miRNA assays. Mouse lung total RNA input ranged from 0.025 to 250 ng per RT reaction. A Caenorhabditis elegans miRNA (miR-2) was included as a negative control assay.

Figure 4

Dynamic range of eight TaqMan miRNA assays using OP9 cell lysates. The number of cell input ranged from 3 to 2500 cells per RT. A Caenorhabditis elegans miRNA (miR-2) was used as a negative control.

Figure 5

Comparison of heat-treated cells, cell lysate and total RNA for real-time quantitation of 10 miRNAs. The level of miRNA expression is measured in the threshold cycles (C_T). Approximately 400 HepG2 cells were analyzed per PCR.

Figure 6

Comparison of TaqMan miRNA miR-16 assay to solution-based northern hybridization analysis. Total RNAs from mouse kidney, liver, lung, spleen and testicle tissues were used.

Figure 7

Discrimination power of let-7 miRNA assays. Relative detection (%) calculated based on C_T difference between perfectly matched and mismatched targets. A total of 1.5 × 10⁸ copies of synthetic RNA was added to RT reaction. The concentration was estimated based on the A₂₆₀ values.

Figure 8

Poor discrimination of miRNAs with solution hybridization-based (northern) analysis.

Figure 9

Specificity of TaqMan miRNA assays between stem–loop and linear RT primers. Mature let-7a-specific assay was tested against let-7a, let-7e and pri-miR precursor let-7a-3. ΔC_T represents the C_T difference between two targets or methods. A total of 1.5 × 10⁸ copies of synthetic targets were added to each RT reaction.