concentration at nuclear speckles cannot be recapitulated with
a Cy5-dye labeled oligonucleotide DNA probe, indicating that
both hybridization-sensitive and nuclease-resistant properties
of ECHO probes are important for effective detection of target
RNA (Fig.3d).- To validate detection specificity, colocalization with protein
markers is used. Protein B23 has been identified as a marker
protein for nucleoli and when it is tagged with DsRed2, its
color separation from D514 RNA probes allows simultaneous
detection of U3 snoRNA (or 28S rRNA) and B23 proteins. - DsRed2-B23 is expressed in the cerebellum from a DNA
expression plasmid using the in vivo electroporation protocol
(Subheading3.2). - Colocalized fluorescence signals indicate target-specific detec-
tion by ECHO-liveFISH (Fig.3e). Likely, these foci are physi-
cally associated with chromatin and nuclear matrix that restricts
their random movement.
3.4.3 Assaying Possible
RNA Interference Caused
by ECHO-liveFISH
siRNA and microRNA target RNA through hybridization, raising
the possibility of probe-induced degradation and translation repres-
sion of the target RNA.- To test this possibility, we perform qRT-PCR to assess the
expression level of the target RNA before and after electropo-
ration of the probes. - Additionally, since 28S rRNA play critical roles in global protein
production, we assess functional interference on 28S rRNA by
measuring gross protein production rate in imaged cells (e.g.,
puromycin labeling as described in SUnSET assay [35]).
äFig. 3(continued) showed high fluorescence background at both intracellular and extracellular locations (D1).
No distinguishable intranuclear structures were resolved by Cy5-d(T) 30 labeling (D2). In contrast, D514-(U) 22
reveals robust fluorescence in nuclei with relatively low background (D3); At higher magnification, D514-(U) 22
reveals readily distinguishable poly(A) nuclear speckles in individual cerebellar cells (D4). (E) Colocalization
between DsRed2-B23 and D514-28S at the nucleoli of electroporated granule neurons. P10 mice expressing
DsRed2-B23 DNA plasmids were perfused and cerebellar slices were processed for DsRed2 and DAPI staining
simultaneously with D514-28S hybridization. Overlapping DsRed2 and D514 fluorescence at nuclear foci
indicates colocalization between B23 proteins and 28S rRNA (white arrowheads). Scale bars: 20μm (B, D1, 3)
and 2.5μm (C, D2, 4, E). Imaging parameter: 405 nm excitation and 425–475 nm detection, 488 nm excitation
and 500–600 nm detection, and 635 nm excitation and 561 nm excitation and 566–703 nm detection were
used to monitor DAPI, EGFP, and DsRed2 fluorescence respectively. Images were acquired in 0.994μm pixel
size and 8μs pixel dwell times. Image stacks of 32.48-μm depth were taken at z-step intervals of 2.32μm. (F)
Time-lapse confocal imaging of poly(A), U3 snoRNA, and 28S rRNA foci in cerebellar granule cells.Top,a
snapshot of nuclear foci imaged in acute cerebellar slices after in vivo electroporation.Bottom,Track-line
presentations of individual fluorescent foci over time (progressing from red to blue, 0–40 minutes). (Panels B,
C, E, and F are reproduced from [24] with permission from Oxford University Press)
Nuclear RNP Dynamics in Mammalian Tissue 269