Journal: Frontiers in Molecular Neuroscience

Article Title: A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons

doi: 10.3389/fnmol.2013.00043

Figure Lengend Snippet: MiRNA profiling by microarray after nucleo-cytoplasmic fractionation of neurons. (A) qRT-PCR analysis of marker genes to validate the fractionation protocol. The fold enrichment (y-axis) of marker genes in the nucleus was calculated by the 2 −dCt [2 −(NUC Ct−CYT Ct) ] method. Bar plots show mean ± standard deviation ( SD ; n = 3). Statistical significance was determined using Student's t -test with Bonferroni correction ( * p < 0.05; ** p < 0.01). (B) Northern blot analysis of the nuclear marker U6 snRNA in nuclear and cytoplasmic fractions. Intensity of the signal was quantified using ImageJ. (C) Detection of nuclear (HDAC2, histone deacetylase 2) and cytoplasmic (beta-Actin) marker proteins in the subcellular fractions using Western blotting assay. Whole cell lysate was used as an input sample. (D) Comparison of different biological replicates from microarray experiments. Pearson's correlation coefficients between indicated samples are shown. Data on gray background represents correlation coefficients for biological replicates from the same cellular fraction. (E) Distribution of miRNA expression in the nucleus and the cytoplasm. Scatterplot of log 2 transformed signal intensity values for miRNAs from nuclear (x-axis) and cytoplasmic (y-axis) fractions (267). Dots above the diagonal indicate cytoplasmic enrichment, below, nuclear enrichment of the respective miRNAs.

Article Snippet: For miRNA profiling analysis, 14 μl of small RNA, obtained from each sample, were sent to microRNA Microarray Service provided by LC Sciences (Texas, USA).

Techniques: Microarray, Fractionation, Quantitative RT-PCR, Marker, Standard Deviation, Northern Blot, Histone Deacetylase Assay, Western Blot, Comparison, Expressing, Transformation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons

doi: 10.3389/fnmol.2013.00043

Figure Lengend Snippet: Comparison of miRNA expression profiles obtained from miRNA microarrays and small RNA deep sequencing. (A) Venn diagram illustrating miRNAs detected by the two different methods. 220 miRNAs were detected by both methods. (B,C) Scatterplot of log 2 transformed signal intensity values (microarray, y-axis) and read counts (deep sequencing, x-axis) for miRNAs detected in the nuclear (B) or cytoplasmic (C) fractions.

Article Snippet: For miRNA profiling analysis, 14 μl of small RNA, obtained from each sample, were sent to microRNA Microarray Service provided by LC Sciences (Texas, USA).

Techniques: Comparison, Expressing, Sequencing, Transformation Assay, Microarray

Journal: Frontiers in Molecular Neuroscience

Article Title: A comprehensive characterization of the nuclear microRNA repertoire of post-mitotic neurons

doi: 10.3389/fnmol.2013.00043

Figure Lengend Snippet: Developmental stage and cell-type-specific expression of the nuclear-enriched miRNAs, miR-25 and miR-92a. (A) Relative expression (normalized to U6 snRNA) levels of miR-25 and miR-92a during in vitro development of primary cortical neurons was determined by qRT-PCR analysis. Bar plots show mean ± SD ( n = 2). Statistical significance was determined using Student's t -test with Bonferroni correction ( * , p < 0.05). (B) Developmental expression score (DES; log 2 (P3/E10) from (Yao et al., ); y-axis) comparison of 10 highest and lowest ranked miRNAs. Error bars represent standard deviation from the mean DES within each group. Statistical significance was determined using Student's t -test ( p = 0.028). (C) Expression of miR-25 and miR-92a in mixed cultures and neuronal-enriched cultures (FUDR-treated). The relative expression levels of indicated RNAs were obtained by the ddCt method. RNA levels in mixed cultures were arbitrarily set to 1. Bar plots show mean ± SD ( n = 3). SD for mixed culture condition was determined after normalization to an internal control RNA (U6 snRNA). Statistical significance was determined based on U6 snRNA normalized values using Student's t -test with Bonferroni correction ( ** p < 0.01). (D) Nuclear-enrichment of miRNA expression in mixed and neuron-enriched (FUDR-treated) cultures. The expression level of miRNAs was determined using qRT-PCR analysis with TaqMan microRNA assay. Bar plots show mean ± SD ( n = 2). Statistical significance was determined using Student's t -test with Bonferroni correction ( * p < 0.05; ** p < 0.01).

Article Snippet: For miRNA profiling analysis, 14 μl of small RNA, obtained from each sample, were sent to microRNA Microarray Service provided by LC Sciences (Texas, USA).

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Comparison, Standard Deviation, Control, TaqMan microRNA Assay