The TaqMan® MicroRNA (miRNA) Assays from Applied Biosystems were developed to detect and quantitatively measure mature miRNA levels in less than four hours from start to finish. There are two-steps: 1) Reverse transcription and 2) Real-Time PCR. The assay requires the use of as little as 1 nanogram of total RNA. Small RNA isolation is not necessary as the assays are designed to only detect the mature miRNA species.
The Reverse transcription step involves the use of a pre-designed looped RT primer to generate first-strand cDNA. This process takes approximately 65 minutes. Once the cDNA is generated, real-time PCR is performed on the samples (approximately two hours). This step utilizes a pre-designed TaqMan miRNA probe, as well as pre-designed forward and reverse primers. The TaqMan probe contains: the FAM reporter dye linked to the 5’ end, a minor groove binder (MGB) to increase the melting temperature of the probe without increasing the length (this is critical since miRNAs are only 22nts and thus probes must be short), and a non-fluorescent quencher (NFQ) at the 3’ end of the probe. Thus, an intact probe containing an NFQ will not fluoresce. During the PCR reaction, the TaqMan probe binds to a sequence in between the forward and reverse primers. During extension the annealed TaqMan probe is displaced and cleaved due to the 5’ end nuclease activity of the polymerase. Cleavage of the reporter dye results in its separation from the NFQ and thus leads to fluorescence. The 3’ end of the probe is blocked, thus the TaqMan probe is not extended during PCR. The fluorescence intensity is measured during each cycle of PCR by the real-time PCR instrument.
I have been using this product quite frequently (weekly) over the last couple of years. The assay is particularly great when samples are limiting, as it requires very little total RNA (1 nanogram) as compared to Northerns and microRNA microarrays, which require microgram amounts. The other benefits of this product are that the assays are extremely sensitive (I have successfully detected down to a few copies of a particular miRNA), rapid (only 3-4 hours as compared to a Northern, which can take several days), reliable (results are consistent), and are very easy to use (no optimization or primer design, just follow the protocol). Additionally, standard Trizol isolated total RNA for these assays can be used. Some of the drawbacks: the reagents are on the expensive side (as compared to doing a Northern), and since the primers are custom made only the canonical mature miRNA sequence can be detected. Thus, the high-specificity of a TaqMan probe may be a double-edged sword, especially if the mature miRNA undergoes editing, as has been reported for some miRNAs. Therefore, another method for detection of a particular miRNA may be necessary to confirm results.