by Harald Lahm and Don Weldon
AL
Screening Live Cells Using RNA Detection Probes
RNA expression is the next frontier in biomarker development because it offers an indication of cellular intent. mRNA expression, in particular, can show how a cell responds to its environ- ment or external factors, and how it reacts to neighboring cells. However, the use of RNA in predicting cellular function has been limited by the availability of suitable tools.
Traditional mRNA detection technologies rely on cell lysis and RNA extraction, which dena- ture the RNA. These technologies typically require laborious sample preparation, nucleic acid purification, reverse transcription (RT) and subsequent analyses based on standard curves. Using cell lysis for mRNA detection is equivalent to studying fossils: it can provide perspective on what the organism was doing at a given time. But the best way to understand a biological organism is to observe it, live, in its natural environment.
Cellular heterogeneity is driving a trend toward single-cell analysis. Even within a given population of homogeneous cell types, differences exist in signaling and expres- sion. The process of establishing induced pluripotent stem cells during reprogram- ming, for example, is not 100% efficient and yields a mixture of cells. Additionally, tumors are extremely heterogeneous, leading to treatment-resistant subpopulations of cells. Therefore, the ability to study mRNA expres- sion in live cells at the single-cell level is vital for research and clinical applications.
Live single-cell RNA detection SmartFlare RNA detection probes from
MilliporeSigma, the U.S. life science business of Merck KGaA (Darmstadt, Germany), enable live-cell RNA detection in a single incubation step using inert nanoparticle technology to
Figure 1 – Cellular uptake of SmartFlare probes is an active process. detect native RNA.1 Researchers can screen
RNA expression in live cells at the single-cell level with minimal to no disturbance or cel- lular stress, permitting downstream analyses using the same unperturbed cells. In addition, the method does not require transfection reagents or target cell manipulation.
Probe development involves the conjugation of a gold nanoparticle to a capture sequence, which is complementary to the target RNA, and a reporter sequence with an attached fluorophore, which is quenched by the gold nanoparticle. Upon binding of the capture se- quence to a target RNA, the reporter is displaced and emits a fluorescence signal. Therefore, the
AMERICAN LABORATORY 32 MARCH 2016
more target RNA present in the cell, the brighter the cells fluoresce.
Nanoparticles enter and leave the cell by en- docytosis and exocytosis, respectively (Figure 1). The probes are simply added to the cell culture medium and endocytosed. If the RNA target is present in the cell, it binds to the capture sequence and releases the now fluo- rescing reporter strand. When the medium is replaced once a day, after a period of about 5–6 days, depending on the cell type, all par- ticles will gradually be exocytosed from the cells. These same cells can then be used for downstream testing.
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