Figure 1.
Scheme of amino-labeled DNA probes for formaldehyde fixation.
(A) Chemical reactions of formaldehyde fixation. (B) Scheme of formaldehyde mediated crosslink between amino-labeled probe and cellular proteins in vicinity. (C) UV-vis absorption spectra of the oligonucleotide probe. Insert: HPLC purification trace of amino-labeled probes. (D) Electrophoretic mobility shift assay (EMSA) of formaldehyde mediated crosslink between the oligonucleotide probes and bovine serum albumin (BSA) on native (Lane 1 to 4) and denaturing PAGE (Lane 5 to 8).
Figure 2.
RNA-DNA FISH of Terra (RNA) and Telomere (DNA) using an amino-labeled oligonucleotide probe.
(A) Either a FITC-labeled PNA probe (green) or an amino-labeled oligonucleotide probe (fluorescently labeled with FAM, green) was used to detect Terra in RNA FISH. Telomere DNA was detected by Cy3-labeled oligonucleotide probes (red) with the sequence of (GGGTTA)6. Nuclei were stained by DAPI (blue). (B) The Terra RNA signal number per nucleus and the signal sizes before and after DNA FISH were compared. A z-series of 3 µm thickness was collected. Images were deconvolved and merged. Individual Terra signals in each cell were computationally recognized by setting a fluorescence intensity threshold. The size of each Terra signal (µm2) was computed. Terra signals measured from one cell are plotted as discrete spots vertically at the x-axis. The P-value was calculated using Student t-test. (C) DNA FISH signal intensity of telomeres was measured to quantitate individual telomere length. The telomeres associated and non-associated with Terra were separated into two groups (n = 100 for each group). The data was presented in box-and-whisker plots. The P-value was calculated using Student t-test.
Figure 3.
RNA-DNA FISH using amino-labeled probes generated by nick translation.
Nuclei were stained by DAPI (blue). Three lncRNAs (Xist, HOTAIR and NEAT2) were detected using Cy3-labeled probes (red). Telomere DNA regions were detected using FITC-labeled oligonucleotide probes (green). Chromosome X territory was detected by FITC-labeled chromosome X paint (green) in DNA FISH. A scale bar of 10 µm is shown.
Figure 4.
RNA-DNA FISH of the single mRNA molecules of EGFP and the telomeres (DNA).
Telomere DNA was detected by Cy3-labeled oligonucleotide probes (shown in red). Nuclei were stained by DAPI (blue). The cells used were mouse fibroblast cells transiently transfected by an EGFP-expressing plasmid. The single mRNA molecules of EGFP were detected by a set of 20 oligonucleotides labeled by Cy5 (shown in grey). Two enlarged images of the single molecule RNA signals are shown on the right side. The images of the single molecule RNA signals are deconvolved single-layer z-sections. A scale bar of 10 µm is shown in each Dapi image. (A) RNA FISH only. (B) RNA-DNA FISH. (C) The total fluorescent signal intensity of the EGFP single mRNA molecule signals per cell (n = 50) measured from the Cy5 channel was normalized by the total EGFP fluorescent intensity per cell measured from the “green” channel. The data was presented in box-and-whisker plots. The P-value was calculated using Student t-test.
Table 1.
Sequences of amino-labeled probe set for single molecule RNA FISH.