Label-Free Fluorescent Copper Nanoclusters for Genotyping of Deletion and Duplication of Duchenne Muscular Dystrophy

Real applications in clinical diagnosis of label-free fluorescent copper nanoclusters (CuNCs) are demonstrated. Double-strand DNA (dsDNA) can act as an effective template for the formation of CuNCs, which can be used to distinguish the deletion or duplication genotypes of Duchenne muscular dystrophy (DMD) due to different fluorescent intensities. After PCR, the DMD amplicons reacted with copper ion by reduction of ascorbic acid and generated fluorescence. The exons of the DMD gene were taken as the model analytes for genetic diagnosis. In this sensing system, the deletion type does not show fluorescence; on the other hand, the duplication type emits higher fluorescence than normal type. Parameters of this sensing system were optimized, including PCR conditions, levels of copper ion and ascorbate, and reaction time. The DMD-dominated exons 45, 46, and 47 were detected, and the method was applied to six samples of DMD patients. The results were consistent with those of the multiplex ligation-dependent probe amplification method. This strategy was feasible to detect all exons of this disease

The testis expresses the highest level of Tra2-β1 among the tissues examined.

<p>Various tissues of 18–20-week-old type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to qRT-PCR to detect <i>Tra2-β1</i> and <i>ASF/SF2</i> mRNA levels. The result showed that among the tissues examined, the testis expressed the highest level of <i>Tra2-β1</i> mRNA, but not the highest level of <i>ASF/SF2</i> mRNA. (B) Proteins were extracted and subjected to Western blotting to detect Tra2-β1 and ASF/SF2 proteins. The result also showed that among the tissues examined, the testis expressed the highest level of Tra2-β1 protein, but not the highest level of ASF/SF2 protein.</p

Overexpression of Tra2-β1, but not ASF/SF2, increases <i>SMN2</i> exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.

<p>Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with <i>SMN2</i> minigene plasmid and <i>Tra2-β1</i> overexpression plasmid, <i>ASF/SF2</i> overexpression plasmid or blank vector as control for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify <i>SMN2</i> minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased <i>SMN2</i> exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with the vector control.</p

The levels of Tra2-β1 and ASF/SF2 decrease during testis cell primary culture, which is correlated with <i>SMN2</i> exon 7 splicing.

<p>(A) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect Tra2-β1, ASF/SF2, hnRNP A1, SRp30c, and hnRNP Q. A representative result of two independent experiments was shown. The result showed that the levels of Tra2-β1, ASF/SF2, and hnRNP A1 decreased dramatically from 2-hour to 96-hour cultures. (B) Total RNA was isolated from primary testis cells cultured for different time periods and subjected to qRT-PCR to detect <i>Tra2-β1</i> and <i>ASF/SF2</i> mRNA expression levels. The result showed that the mRNA levels of <i>Tra2-β1</i> and <i>ASF/SF2</i> decreased after longer cultures, which was consistent with the protein expression. Error bars represent standard deviation. (*) <i>P</i> < 0.01, compared with 2-hour cultured cells.</p

The testis of SMA mice expresses high levels of <i>SMN2</i> full-length mRNA and protein.

<p>Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both <i>SMN2</i> full-length (FL) and exon 7-lacking (Δ7) mRNAs. A representative result of six independent experiments was shown. The result showed that the testis expressed high level of <i>SMN2</i> FL mRNA. (B) Proteins were extracted and subjected to Western blotting to detect SMN protein expression. The result showed that SMA mouse testis expressed the highest level of SMN protein among the tissues examined.</p

The levels of <i>SMN2</i> full-length mRNA and protein decrease during testis cell primary culture.

<p>(A) Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours and 24 days) and subjected to RT-PCR to amplify <i>SMN2</i> FL and Δ7 mRNAs. For comparison, total RNA isolated directly from the testis and liver was also analyzed. A representative result of three independent experiments was shown. The result showed that primary testis cells after a 2-hour culture still expressed high level of <i>SMN2</i> FL mRNA. However, the level decreased after longer cultures. (B) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect SMN protein. The result showed that SMN protein level was high in 2-hour cultured cells and decreased dramatically in 96-hour cultured cells, consistent with the result of <i>SMN2</i> exon 7 splicing. (C) snRNP complexes were isolated from primary testis cells cultured for 2 hours and 96 hours by anti-Sm antibodies. Various snRNAs were then extracted and quantitated by real-time PCR. The result showed that the levels of snRNP complexes decreased in 96-hour cultured cells compared with 2-hour cultured cells. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with 2-hour cultured cells.</p

Knockdown of Tra2-β1 decreases <i>SMN2</i> exon 7 inclusion in primary testis cells of SMA mice.

<p>Primary testis cells of SMA mice were cultured for 72 hours and then transfected with Tra2-β1 siRNA or negative control (NC) siRNA for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify <i>SMN2</i> FL and Δ7 mRNAs. The results show that knockdown of Tra2-β1 promoted <i>SMN2</i> exon 7 exclusion in primary testis cells of SMA mice. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with the siNC control.</p

Severe denervation of FDB-2muscle in severe SMA mouse is restored by MO treatment.

<p>(A-C) The confocal images are Z-stack projection images at P9, stained with anti-synaptophysin antibody, anti-neurofilament antibody, and conjugated α-bungarotoxin protein from FDB-2 muscles of control, severe SMA, and MO-treated severe SMA mice. The images in parallel show individual channels of grey-scale images and merged images. (A) FDB-2 muscle from control mice (n = 3). NMJs are partially or fully innervated. (B) FDB-2 muscle from severe SMA mice (n = 3). Most NMJs are fully denervated (arrow). (C) FDB-2 muscle from severe SMA mice with MO treatment. Recovery is observed after MO treatment by SC injection at P0. (D) The endplate size of AT muscle in severe SMA mice, MO-treated SMA mice, and control mice at P9. (E) Quantification of fully innervated endplates (blue), partially denervated endplates (yellow), and fully denervated endplates (red) in FDB-2 muscles of control, severe SMA, and MO-treated severe SMA mice at P9. One hundred of individual muscles were counted in each mouse, and 3 pairs of control, severe SMA and MO-treated severe SMA mice were studied. All quantitative data are mean ± SEM, ***<i>P</i> < 0.001.</p

NMJ denervation in different pattern muscles of severe SMA mice at P9.

<p>(A) Quantification of fully innervated endplates in muscles of control (blue) and severe SMA (red) mice at end stage (P9). (B) Quantification of fully innervated endplates (blue), partially denervated endplates (yellow), and fully denervated endplates (red) in muscles of severe SMA mice at P9. One hundred NMJs of individual muscles were counted in each mouse, 3 pairs of severe SMA and control mice were studied. All quantitative data are mean ± SEM. (FDB, flexor digitorum brevis; AT, anterior tibialis; EDL, extensor digitorum longus; SPI, serratus posterior inferior)</p

The full denervation of endplates is observed on the FDB-2 muscle of severe SMA mice at P9.

<p>The confocal images are Z-stack projection images, stained with anti-synaptophysin and anti-neurofilament antibodies and conjugated α-BTX protein, in control and Taiwanese severe SMA mice at P9. The images in parallel show individual channel grey-scale images and merge images. (A, B) FDB-2 muscle from control (n = 3) and severe SMA mice (n = 3). Most NMJs are fully denervated (arrow) or partially innervated in the FDB-2 muscle of severe SMA mice. (C, D) SPI muscles from control mice and severe SMA mice, (E, F) splenius capitis muscles from control mice and severe SMA mice, and (G, H) longissimus capitis muscles from control mice and severe SMA mice show little difference. (FDB, flexor digitorum brevis; SPI, serratus posterior inferior; α-BTX, α-bungarotoxin).</p