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 environment 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 denature 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 expression. The process of establishing induced pluripotent stem cells during reprogramming, 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 expression 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 EMD Millipore (Billerica, Mass.) enable live-cell RNA detection in a single incubation step using inert nanoparticle technology to detect native RNA.1 Researchers can screen RNA expression in live cells at the single-cell level with minimal to no disturbance or cellular 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 sequence to a target RNA, the reporter is displaced and emits a fluorescence signal. Therefore, the more target RNA present in the cell, the brighter the cells fluoresce.

Nanoparticles enter and leave the cell by endocytosis 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 fluorescing reporter strand. When the medium is replaced once a day, after a period of about 5‒6 days, depending on the cell type, all particles will gradually be exocytosed from the cells. These same cells can then be used for downstream testing.

Figure 1 ‒ Cellular uptake of SmartFlare probes is an active process.

SmartFlare probes are nontoxic and do not alter gene expression, translation or cell proliferation (Figure 2). The lyophilized probes are reconstituted with sterile nuclease-free water, diluted with phosphate-buffered saline (PBS) to the desired concentration, added to the cell culture medium and incubated with the cells overnight. RNA levels are typically detected using fluorescence microscopy or flow cytometry the next day.

Figure 2 ‒ SmartFlare probes are nontoxic and do not alter gene expression.

Uptake, scramble and housekeeping control probes

When screening live cells for RNA detection, it is important to include three experimental control probes: uptake, scramble and housekeeping. The uptake control is always fluorescing and helps determine the cell’s ability to internalize the probes. It is important to use because not all cells will internalize the probes equally.

The scramble probe serves as a background control. Because the “reporter” sequence attached to the scramble control has no counterpart in the genome of mammalian or eukaryotic cells, any fluorescence released from the scramble control is nonspecific (e.g., probe degradation, incomplete quenching) and can serve as the background fluorescence. The housekeeping gene probe (e.g., GAPDH, β-actin) acts as a positive control that the SmartFlare probes can detect specific gene expression in a given cell population.

Heterogeneous cancer cell populations

The heterogeneity of cancer cells is a significant barrier to developing and receiving effective treatments. When studying mRNA expression levels in heterogeneous populations of cells, significant differences in data are expected if RNA is analyzed at the population level versus the single-cell level.

Quantitative RT-PCR cannot distinguish between high- and low-expressing subpopulations in heterogeneous cell samples. MCF-7 and MDA-MB-231 breast cancer cells have no/low and high expression levels of IL-6, respectively. The RNA expression profile changes accordingly when different percentages of these cells are mixed. While analyzing unknown heterogeneous cell populations, the researcher does not know whether variations in the curve represent a small subpopulation of cells changing expression to a very high degree, or if all the cells are changing expression to a very low degree collectively. Use of SmartFlare probes resolves expression at the single-cell level, allowing for the identification by flow cytometry of divergent cell populations not detectable by qRT-PCR. Therefore, single-cell resolution of mRNA expression is a powerful tool for studying heterogeneous cell populations.

Monocyte differentiation

Live-cell mRNA detection is also valuable for studying cell differentiation over time. THP-1 monocytes highly express c-MYC mRNA. These cells were treated with phorbol myristate acetate (PMA) to induce their differentiation to macrophages, and c-MYC expression was analyzed by quantitative reverse transcription (qRT)-PCR and live-cell RNA detection via SmartFlare probes at days 0 and 5, respectively. As expected, c-MYC mRNA expression decreased dramatically during monocyte differentiation by day 5, the time it takes for these cells to differentiate (Figure 3a).

Figure 3 ‒ a) c-MYC mRNA expression decreases during monocyte differentiation. b) Monitoring mRNA expression and morphology change over time. c) c-MYC mRNA expression changes during differentiation are independent of nanoparticle uptake values.

Cellular morphology and RNA expression can be observed simultaneously and monitored over time using the probes (Figure 3b). However, researchers must also consider the possibility that the cell is continuing to express mRNA but no longer internalizing the nanoparticle upon differentiation. The uptake control probes demonstrate that there is no change in the uptake of the nanoparticle five days post-PMA treatment, which confirms that c-MYC mRNA levels decrease in THP-1 cells during differentiation (Figure 3c).

Pluripotent stem cells

The successful creation of induced pluripotent stem (iPS) cells is important for research and clinical applications. However, identifying truly reprogrammed iPS cells can be difficult and often requires fixation and thus sacrifice of the cells. SmartFlare RNA detection probes were able to detect pluripotency gene expression in live embryonic and iPS cells.2 Probes specific for GAPDH, NANOG and GDF3 detected gene expression in iPS cells across three species: human, mouse and pig. A parallel qRT-PCR analysis was conducted to confirm the expression of these genes in iPS cells of all three organisms. Application of the probes did not affect either protein or mRNA expression and had no effect on cell proliferation.

The probes were used as a live screening tool to identify reprogrammed murine iPS cells derived from murine tail-tip fibroblasts in situ based on their fluorescence intensity. Murine tail-tip fibroblasts were transduced with the murine STEMCCA vector (EMD Millipore), which contains all four classical Yamanaka reprogramming factors. Doxycycline was added from day 2 on, and the cells were seeded onto a murine embryonic fibroblast feeder layer on day 3. By day 9, colonies had formed, and probes specific for either NANOG or GDF3 were added. On day 10, individual colonies with either bright or faint fluorescence were identified under the microscope, replated on day 11 and expanded. After the second passage of these cells, pluripotency gene expression was evaluated by qRT-PCR.

The data demonstrated that NANOG is a reliable marker for the identification of truly reprogrammed iPS cells. This conclusion was confirmed upon further evaluation in downstream differentiation experiments using murine embryonic stem cells as a control.3 NANOG-selected colonies expressed markers specific for endoderm, ectoderm or cardiac mesoderm at levels comparable to that of differentiated murine embryonic stem cells. Additionally, these clones allowed the identification of cardiac progenitors based on their green fluorescent protein (GFP) fluorescence. Colonies with a high NANOG-specific fluorescence generated a strongly increased fraction of cardiac progenitors upon differentiation. Therefore, NANOG-specific SmartFlare probes can be used to identify truly reprogrammed developing murine iPS cell colonies live and in situ.

Conclusion

SmartFlare RNA detection probes can be used in a variety of cell types to reliably detect the expression of mRNA and miRNA in single live cells without manipulation of the target cell. The probes are nontoxic, do not affect mRNA or protein expression and do not impact cell proliferation. Importantly, the same treated cells can be used to study functionality in downstream experiments. This technology can be particularly powerful for studying heterogeneous cell populations and cell differentiation over time, identifying reprogrammed iPS cells and for functional assessment of RNA biomarkers.

References

  1. Seferos, D.S.; Giljohann. D.A. et al. Nano-flares: probes for transfection and mRNA detection in living cells. J. Am. Chem. Soc. 2007, 129(50), 15,477‒9.
  2. Lahm, H.; Doppler, S. et al. Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent cells of different species. Stem Cells 2015, 33(2), 392‒402; doi: 10.1002/stem.1872.
  3. Wu, S.M.; Fujiwara, Y. et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 2006 Dec 15, 127(6), 1137‒50.

Harald Lahm, Ph.D., is Head of Laboratory of Molecular and Cell Biology of Experimental Surgery, German Heart Center Munich. Don Weldon is R&D Manager, EMD Millipore Corp., 290 Concord Rd., Billerica, Mass. 01821, U.S.A.; tel.: 951-514-4566; e-mail: [email protected]; www.emdmillipore.com. This article is based on Ref. 2 and a GenomeWeb webinar presented by Harald Lahm and Don Weldon.

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