Subsequently, we validated the MIWI/piRNA-mediated repression of target mRNAs with reporter-based assays and showed that both the slicer activity and the piRNA-loading ability of MIWI are critical for the piRNA-mediated target repression.
When we exposed the slices to Aβ 25-355) before the separation and measured the MTT reduction activity two hours after the preparation, there was no effect of Aβ 25-355) on the MTT reduction of both, the slices and the single cells (Figure 3A).
This results from both the slice-selective excitation and the need to acquire several scans to construct the complete FID.
These data unequivocally demonstrate that both the slicer activity and the piRNA-loading ability of MIWI/HIWI are indispensable for piRNA-induced target repression.
Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression.
In the case of tomato leaf abscission, cell separation and GUS expression occurred in both the slices that were tied back onto the AZ or placed on agar (Fig. 7).
Our reporter assays further demonstrated that piRNAs could induce downregulation of their targets, and both the slicer activity and piRNA-loading ability of MIWI are required for such piRNA-guided target repression.
The device provides a stable microfluidic perfusion environment in which tissue health is optimized by superfusing both surfaces of the slice.
We suggest three ways to improve the oxygen supply to slices under submerged conditions: (i) optimizing chamber design for laminar flow of superfusion fluid; (ii) increasing the flow rate of superfusion fluid; and (iii) superfusing both surfaces of the slice.
