Unlocking the mysteries of miRNA

Through the years, miRNA has proven difficult to detect due to its small size and low abundance. There are three methods commonly used in detecting and quantifying miRNA. The first relies on next-generation sequencing (NGS), which generates sequence information for all miRNAs present in a sample. While this makes NGS useful for discovering novel miRNA variants, the technique is costly and requires large amounts of input material. Furthermore, NGS cannot accurately measure miRNA levels.

Another technique is the microarray, which compares changes in miRNA expression. Microarrays rely on the binding of miRNA to fluorescent probes, with the resulting optical signal indicating the relative quantity of DNA. The brighter the signal, the more there is of a specific miRNA. Like NGS, however, microarrays are not quantitative, making them sometimes unable to detect very low levels of miRNA. 

Unlike the two previous methods, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has the ability to quantify the amount of miRNA in a given sample. MiRXES’ miRNA-based diagnostic products rely on RT-qPCR, albeit with a unique twist that makes our technology far more sensitive than the rest. 

As easy as one, two, three (primers)

MiRXES’ patented RT-qPCR technology relies on the three-primer approach. The first kind of primer comes into play during the initial step of RT-qPCR, called reverse transcription. In this step, the enzyme reverse transcriptase converts miRNA into complementary sequences of DNA. This is made possible by using conformation-restricted primers designed specifically for each miRNA. These primers are distinguished by their stem loop structures, which prevent the primers from detaching easily once it binds to the miRNA.

After reverse transcription, the resulting DNA is amplified with the help of forward and reverse primers that attach on either side of the sequence, further ensuring specificity. With each cycle of PCR, the amount of DNA in the sample exponentially multiplies until it reaches a detectable level.

Finally, to quantify the DNA (and by extension, the target miRNA), PCR progress is tracked by the machine in real-time using dyes that nestle in between the DNA bases. As each dye corresponds to a distinct sequence, multiple miRNA can be detected and quantified even in a single set-up. Collectively, these three primers guarantee the sensitive, specific and robust detection of miRNA in biological samples.