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Results: 4

1.
Fig. 1.

Fig. 1. From: Tailor-made RNAi knockdown against triplet repeat disease-causing alleles.

Schematic view of the pull-down method. cDNAs prepared from patients’ RNAs (1) were subjected to hybridization with the biotin-CAG RNA probe (2); and the hybridized cDNAs were recovered by pull-down with avidin-resin (3, 4), followed by elution (5). The recovered cDNAs were examined by the TaqMan SNP Genotyping assay (6). When SNP sites were heterozygous, the SNP typing with the recovered cDNAs plotted the spots distant from the standard curves.

Masaki Takahashi, et al. Proc Natl Acad Sci U S A. 2010 December 14;107(50):21731-21736.
2.
Fig. 4.

Fig. 4. From: Tailor-made RNAi knockdown against triplet repeat disease-causing alleles.

Disease allele-specific RNAi knockdown against the mutant HTT alleles carrying distinct cSNP haplotypes. (A) ASP-RNAi knockdown by siRs099_T10 and siRs099_C9(G14) siRNAs, which targeted the T and C alleles, respectively, at the rs363099 cSNP site in HTT were examined by IC50 analysis: the effects of the designed siRNAs on suppression of target alleles and allele discrimination were investigated. Data are the average of four independent determinations. Error bars represent SDs. (B) Western blot analyses of the endogenous HTT protein. On the basis of the C0142 and C0221 cSNP haplotypes (Fig. 2C), appropriate siRNA targeting the rs363099 cSNP site in the respective mutant HTT alleles was chosen and introduced into lymphoblastoid cells from the corresponding HD patient. After 3-d incubation, cell lysate was prepared and examined by Western blotting with anti-human HTT antibody; anti-α-tubulin antibody was used as an internal loading control. The siRNAs and lymphoblastoid cell lines are indicated above each gel. A healthy individual (N0001) was also examined as a control. The mutant and normal HTT isoforms are indicated by M and N, respectively.

Masaki Takahashi, et al. Proc Natl Acad Sci U S A. 2010 December 14;107(50):21731-21736.
3.
Fig. 2.

Fig. 2. From: Tailor-made RNAi knockdown against triplet repeat disease-causing alleles.

Biased recovery of disease-causing HTT alleles. (A) cDNAs prepared from two HD cases (C0142 and C0221) were subjected to the pull-down method (+), and then the length of the CAG trinucleotide repeat array in HTT was investigated by PCR analysis. Untreated cDNAs (−) were also examined as a control. (B) cDNAs (C0142 and C0221), which were the same as in A, were subjected to the TaqMan SNP Genotyping assay, and the results were plotted in blue. Standard curves (indicated in black) were constructed by the same SNP typing using serially diluted (0.01, 1, 10 ng) untreated cDNAs. The examined coding SNP (cSNP) sites are indicated. x- and y-axes indicate relative fluorescent intensity obtained from the TaqMan probes against the indicated alleles, respectively, and allelic nucleotides at the cSNP sites are also shown in parentheses. (C) Linkage between the expanded CAG trinucleotide repeats and cSNPs was estimated in each HD case according to the results of the cSNP typing in B. (D) Confirmatory experiments by conventional methods. The same SNP typing as in B was carried out using the isolated plasmids carrying the HTT alleles, which were obtained by conventional methods (Fig. S2). Dots in blue and open circles represent the results of the plasmids containing the mutant and normal HTT alleles, respectively. Indicated clone (plasmid) numbers correspond to those indicated in Fig. S2.

Masaki Takahashi, et al. Proc Natl Acad Sci U S A. 2010 December 14;107(50):21731-21736.
4.
Fig. 3.

Fig. 3. From: Tailor-made RNAi knockdown against triplet repeat disease-causing alleles.

Pull-down of (A) ATXN3, (B) CACNA1A, and (C) ATN1 cDNAs carrying longer CAG trinucleotide repeats. cDNAs were prepared from indicated human cell lines and treated by the pull-down method. The recovered cDNAs were examined by PCR analysis and assessed CAG trinucleotide repeat array length in each gene as in Fig. 2A. Because the CAG trinucleotide repeat arrays in ATXN3 (A) were homozygous in all of the samples investigated, mixtures of the HEK293 and HeLa cDNAs (Sample 4) and of the HeLa and SH-SY5Y cDNAs (Sample 5) were prepared and then examined. (D and E) Allele discrimination capability of the pull-down method was examined. The Alexa 488- and Alexa 647-labeled CTG oligonucleotides with various numbers of the CTG trinucleotide repeats, which are complementary to the biotin-CAG RNA probe, were used as targets. The number (n) of the CTG repeats is indicated as (CTG)n. (CTG)20 oligonucleotides, the longest CTG repeats arrays (20 repeats), were labeled with Alexa 488 (D) or Alexa 647 (E) and individually mixed with a competitor, a (CTG)n oligonucleotide labeled with the other fluorescent dye; these mixtures were then subjected to the pull-down assay. The fluorescence intensities of the oligonucleotides recovered by the pull-down method were examined. Ratios of normalized fluorescent intensities of the competitors to that of the (CTG)20 oligonucleotide are shown. Data are averages of six independent tests. Error bars indicate SD. P values were calculated by Dunnett's multiple comparison test.

Masaki Takahashi, et al. Proc Natl Acad Sci U S A. 2010 December 14;107(50):21731-21736.

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