RNA DETECTION PROBES continued
Monocyte differentiation Live-cell mRNA detection is also valuable for
Figure 2 – SmartFlare probes are nontoxic and do not alter gene expression.
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 transcrip- tion (qRT)-PCR and live-cell RNA detection via SmartFlare probes at days 0 and 5, respec- tively. As expected, c-MYC mRNA expression decreased dramatically during monocyte dif- ferentiation by day 5, the time it takes for these cells to differentiate (Figure 3a).
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 dem- onstrate that there is no change in the uptake of the nanoparticle five days post-PMA treat- ment, 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 anal- ysis 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.
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.
AMERICAN LABORATORY 34 MARCH 2016
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 (MilliporeSigma), which contains all four classical Yamanaka
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