one-way anova with dunnett’s post hoc multiple comparisons tests (graphpad prism 6 software)  (GraphPad Software Inc)

Journal: Nucleic Acid Therapeutics

Article Title: Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides

doi: 10.1089/nat.2022.0046

Figure Lengend Snippet: Effect of LNA content and ON lengths on the efficiency of downregulating HTT mRNA. (A) HTT mRNA levels 4 days after transfection of GM04281 human HD fibroblasts carrying 68 repeats on the disease allele with ON (100 nM). (B) Percentage of LNA and calculated T m in the different ONs used. (C) HTT mRNA levels 4 days after transfection of GM04281 human HD fibroblasts with long ONs having a different backbone (100 nM). Error bars = SD ( n ≥ 3), n.s., nonsignificant, * P ≤ 0.05, **** P ≤ 0.0001 (one-way ANOVA, post hoc Bonferroni). ANOVA, analysis of variance; HTT , Huntingtin gene; SD, standard deviation T m , melting temperature.

Article Snippet: Statistical significance was determined by one or two-way analysis of variance (ANOVA) followed by individual comparisons using the Bonferroni test (GraphPad Prism 6 Software; GraphPad Software, Inc.).

Techniques: Transfection, Standard Deviation

Journal: Nucleic Acid Therapeutics

Article Title: Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides

doi: 10.1089/nat.2022.0046

Figure Lengend Snippet: Evaluating the efficacy of different palmitoyl-conjugated anti-gene ONs in downregulating HTT mRNA. (A) HTT mRNA levels 4 days after transfection (100 nM) of various PS backboned 16-mer ONs (sequences in ) into GM04281 human HD fibroblasts carrying 68 repeats on the disease allele. (B) HTT mRNA levels 4 days after transfection (100 nM) of PO backboned 16-mer ONs (sequences in ) into GM04281 human HD fibroblasts. Irr pal (PS/PO) sequences have the palmitoyl-modified LNA units positioned similarly as in Pal 2 × 3′ ONs. Error bars = SD ( n ≥ 3), ** P ≤ 0.01 (one-way ANOVA, post hoc Bonferroni). PO, phosphodiester; PS, phosphorothioate.

Article Snippet: Statistical significance was determined by one or two-way analysis of variance (ANOVA) followed by individual comparisons using the Bonferroni test (GraphPad Prism 6 Software; GraphPad Software, Inc.).

Techniques: Transfection, Modification

Journal: Nucleic Acid Therapeutics

Article Title: Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides

doi: 10.1089/nat.2022.0046

Figure Lengend Snippet: Evaluation of the efficacy of the lead ON candidates under different experimental settings. (A) HTT mRNA levels 4 days after transfection (involving serum starvation conditions for 4 h) of selected ON candidates with PS backbone (sequences in ) into GM04281 human HD fibroblasts carrying 68 repeats on the disease allele (final concentration 100 nM). (B) HTT mRNA levels 4 days after transfection (100 nM) of selected long ON candidates with PS backbone (sequences in ) into human HD fibroblasts with different repeat lengths (the numbers between brackets refer to the number of repeats of mutant and wild-type HTT CAG•CTG trinucleotide repeats). Error bars = SD ( n ≥ 3), n.s., nonsignificant, * P ≤ 0.05, ** P ≤ 0.01 (one or two-way ANOVA, post hoc Bonferroni). Red asterick represent the statistical significance of Pal 2 × 3′ PS vs CAG 16 PS effect under serum-free or serum-associated transfection conditions.

Article Snippet: Statistical significance was determined by one or two-way analysis of variance (ANOVA) followed by individual comparisons using the Bonferroni test (GraphPad Prism 6 Software; GraphPad Software, Inc.).

Techniques: Transfection, Concentration Assay, Mutagenesis

Journal: Nucleic Acid Therapeutics

Article Title: Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides

doi: 10.1089/nat.2022.0046

Figure Lengend Snippet: Effects of anti-gene CAG ONs on HTT protein levels in GM04281 human HD fibroblasts. (A, B) Representative gels for western blots performed with selected ON candidates. The top two bands correspond to HTT protein (upper band mutant; lower band wild-type allele). The band in the bottom marks importin 7 protein and used as a loading control. (C, D) HTT protein level quantified by ImageJ and normalized to importin 7 after 4 days of transfection of GM04281 human HD fibroblasts carrying 68 repeats on the disease allele (final concentration 100 nM) with different 16 mer CAG ONs with PS backbone and long CAG ON with different backbones, respectively. Error bars = SD ( n ≥ 3), n.s., nonsignificant, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 (one-way ANOVA, post hoc Bonferroni).

Article Snippet: Statistical significance was determined by one or two-way analysis of variance (ANOVA) followed by individual comparisons using the Bonferroni test (GraphPad Prism 6 Software; GraphPad Software, Inc.).

Techniques: Western Blot, Mutagenesis, Control, Transfection, Concentration Assay