Table 1.
List of oligonucleotide primers, with linker sequence underlined, used for qRT-PCR experiments.
Fig 1.
Mutant sense DMPK RNA nuclear foci in human DM1 fetal samples.
RNA foci containing (CUG)n expansion were labeled in red, using a 5’-Cy3-labeled (CAG)5 PNA probe in heart, skeletal muscle and brain samples from 12–13.5 week-old (12–13.5 wk), 16–17 week-old (16–17 wk) and 33–33.5 week-old (33–33.5 wk) DM1 fetuses.
Fig 2.
Mutant sense DMPK RNA nuclear foci in DMSXL embryos and neonates.
RNA foci containing (CUG)n expansion were labeled in red, using a 5’-Cy3-labeled (CAG)5 PNA probe in E14.5 embryos heart, muscle (hind leg) and brain (cortex) and P7 neonates heart (ventricule), muscle (gastrocnemius) and brain (frontal cortex).
Fig 3.
Quantification of mutant sense DMPK RNA nuclear foci intensity in DMSXL embryos and neonates.
Intensity was measured in three E14.5 embryos heart, muscle (hind leg) and brain (cortex) and three P7 neonates heart (ventricule), muscle (gastrocnemius) and brain (frontal cortex). The graph represents the mean of the normalized intensity (total intensity/number of nuclei in arbitrary units, a.u.) ± SEM.
Fig 4.
Co-localization of the mutant sense DMPK RNA nuclear foci with MBNL1 and MBNL2 proteins.
In situ hybridization was combined with immunofluorescence in skeletal muscle tissues from a human DM1 13.5 week-old fetus (A) and from a DMSXL E14.5 muscle hind leg (B). Left panels show hybridization with a red fluorescent probe recognizing expanded (CUG)n sense DMPK RNA transcripts, middle panels show immunofluorescence using MBNL1 or MBNL2 antibodies and right panels merged images showing RNA and protein co-localization.
Fig 5.
Mutant antisense DMPK RNA nuclear foci in human DM1 fetal samples.
RNA foci containing (CAG)n expansions were labeled in red, using a 5’-Cy3-labeled (CTG)5 PNA probe in heart, skeletal muscle and brain samples from 12–13.5 week-old (12–13.5 wk), 16–17 week-old (16–17 wk) and 33–33.5 week-old (33–33.5 wk) DM1 fetuses.
Fig 6.
Mutant antisense DMPK RNA nuclear foci in DMSXL embryos and neonates.
RNA foci containing (CAG)n expansions were labeled in red, using a 5’-Cy3-labeled (CTG)5 PNA probe in E14.5 embryos heart, muscle (hind leg) and brain (cortex) and P7 neonates heart (ventricule), muscle (gastrocnemius) and brain (frontal cortex).
Fig 7.
Simultaneous detection of mutant sense and antisense DMPK RNA nuclear foci.
Localization of sense and antisense DMPK foci was studied using 5’-cy3-(CAG)5 (recognizing sense (CUG)n transcripts in red) and 5’-Alexa 488-(CTG)5 (recognizing antisense (CAG)n transcripts in green) probes in the same experiment. Left panel: human 12 week-old fetus heart sample; right panel: DMSXL E14.5 embryonic muscle from the hind leg.
Fig 8.
Sense and antisense DMPK RNA levels in DM1 fetuses.
qRT-PCR were performed in heart (left panels) and in brain (right panels) using specific primers and standard curves established with a known number of plasmid molecules with the amplicons as described previously [32]. Levels of sense and antisense DMPK transcripts were reported on graphs using 18S as internal control, in arbitrary units (a.u.). Cont 18–25: samples from control fetuses aged between 18–25 weeks. DM1 16–20: samples from DM1 fetuses aged between 16–20 weeks. DM1 33: sample from a 33 weeks old DM1 fetus. The mean and SEM are represented respectively, as horizontal and vertical bars. (**, p<0.01, two-tailed Student’s t-test).
Fig 9.
Sense and antisense DMPK RNA levels in transgenic mice.
qRT-PCR were performed in heart, skeletal muscle and brain from DMSXL and DM20 embryos and neonates at embryonic E14.5 to postnatal P29 stages. Levels of sense DMPK (left panels) and antisense transcripts (right panels) were reported on graphs using 18S as internal control, in arbitrary units (a.u.) with standard deviation of the mean for repeated experiments.