Profiling Single Cells
The ability to create an miRNA profile from a single cell has major ramifications, particularly with neuronal and stem cell research, according to Caifu Chen, Ph.D., director of assays R&D at Applied Biosystems (www.appliedbiosystems.com).
“Our company made a major breakthrough in single-cell gene-expression analysis,” he reported. “We developed RNA-profiling technologies that need only miniscule amounts of RNA for very reliable, reproducible, and quantitative PCR.”
The Multiplex RT for TaqMan® MicroRNA Assays allows profiling of both miRNAs and mRNAs from the same cell. “What makes this process unique and possible is a multiplex preamplification step that amplifies cDNA with primer sets before specific target amplification. This gives researchers the needed sensitivity to work with extremely small samples such as single cells. An added benefit is that this does not introduce amplification bias,” Dr. Chen noted.
How does it work? A three-step protocol enables researchers to quantify only mature miRNAs. A multiplex reverse transcription (RT) reaction with a pool of approximatley 450 stem-loop RT primers is used to convert RNA to cDNA. Then, the cDNA product is subjected to multiplex preamplification. Finally, individual TaqMan reactions are performed using arrays of TaqMan MicroRNA Assays in 384-well plates that accurately determine the level of miRNA expression.
Another application of the technology is the generation of miRNA profiles from biopsy material for clinical research. “Often one has only a small amount of genetic material from patient biopsies. Being able to create miRNA profiles is significant for these kinds of applications. Importantly, our protocol is also suitable for formalin-fixed, paraffin-embedded samples,” added Dr. Chen.
He feels that real-time PCR is better suited for capturing miRNA-expression information as compared to microarrays. “Because of the increased assay sensitivity, you can start with 10-fold less RNA and still detect miRNAs expressed at a lower level. In other words, TaqMan miRNA assays will help researchers see more miRNAs,” Dr. Chen explained.
Other advantages he highlighted for the technology include a broader dynamic range that benefits the widely varying levels of miRNA, greater specificity for discriminating closely related miRNAs, and reduced sample consumption.