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In addition to examining the RPC profiles for the expression of classical RPC marker genes, we wished to identify new genes that were consistently expressed among most or all RPCs, and thus could serve as new markers of RPCs and perhaps reveal new findings regarding RPC biology.
This finding corroborates the microarray results, where not all RPCs were positive for these RPC marker genes (see Figure 2A).
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The real-time PCR analysis showed that the expression of the RPC markers PAX2, WT1, Six2 and CD24 increased after RAB treatment (Fig. 1a), indicating that RPCs were successfully induced.
To investigate the heterogeneous expression of the newly identified broadly expressed RPC markers, section ISH and DISH were performed for one such gene, Fgf15.
At the same time, they also show that a clear minority of [3H]-thymidine+ cells was negative for these RPC markers.
In addition to the validation of previously characterized RPC markers, the single cell method revealed the expression of new genes broadly expressed in RPCs that were not previously characterized in the retina, such as tropomyosin 4 (Tpm4).
As expected, the expression of the RPCs markers was further up-regulated compared with that of the RAB group, as shown by real-time PCR analysis after 5 days of differentiation in RAB medium (Fig. 1a).
In addition to this insight into cell cycle function, this observation means that Kpna2 can be used as a marker for RPCs in S phase in future studies.
To accomplish this, a post hoc classification scheme was devised based upon clusters of co-expressed genes that were centered around previously identified markers of RPCs, RGCs, ACs or rod photoreceptors.
98 Additionally, Mash1 is expressed by a subset (10%30%%, depending on age) of the total proliferating progenitor cells, providing a molecular marker of heterogeneity among retinal progenitor cells (RPCs).
Only 2/6 of the RPCs denoted as transitional cells scored highly for G2/M markers (Figure 13), suggesting that these cells are most likely in different windows of the transition from RPC to postmitotic neuron.
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