14 research outputs found
Functional validation of a miRNA-34a-TREM2–3’UTR interaction.
<p><b>(A)</b> ribonucleotide sequence of the 299 nt TREM2-mRNA-3’-UTR is shown in the 5’-3’ direction; the 22 nt miRNA-34a-TREM2 3’UTR complementarity-interaction region is indicated by a black underline and the 8 nt TREM2-mRNA-3’-UTR seed sequence <b>5’-ACACUGCU-3’</b> is overlaid in yellow; a single arrowhead indicates the 5’ end of a poly A+ tail in the TREM2 mRNA (22 ‘A’ nt shown; the length of this poly A+ tail is variable); TREM2 mRNA sequence derived from NM_018965; TREM2 transcript is the major X1 variant (see also <a href="http://switchdb.switchgeargenomics.com/productinfo/id_801321/" target="_blank">http://switchdb.switchgeargenomics.com/productinfo/id_801321/</a>) (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.g003" target="_blank">Fig 3</a></b>); <b>(B)</b> TREM2-mRNA-3’UTR expression vector luciferase reporter assay (pLightSwitch-3’UTR; Cat#S801178; Switchgear Genomics, Palo Alto CA); in this vector, the entire 299 nucleotide TREM2 3’UTR was ligated into the unique Nhe1-Xho1 site; <b>(C)</b> control C8B4 murine microglial cells, 1 week in culture; phase contrast bright field microscopy 20x; C8B4 cells transfected with the TREM2-mRNA-3’UTR expression vector luciferase reporter were treated exogenously with miRNA-34a, a scrambled control miRNA-34a (miRNA-sc) or control miRNA-183; see references and text for further details [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref019" target="_blank">19</a>]; <b>(D)</b> compared to control, C8B4 cells transfected with a scrambled (<b>sc</b>) control pLightSwitch-3’UTR vector, the TREM2-mRNA-3‘UTR vector exhibited decreased luciferase signal to a mean of 0.16-fold of controls in the presence of miRNA-34a; this same vector exhibited no change in the presence of the control miRNA-34a-sc or miRNA-183; for each experiment (using different batches of MG cells) a control luciferase signal was generated and included separate appropriate controls with each analysis; in addition a control vector β-actin-3’UTR showed no significant effects on the relative luciferase signal yield after treatment with either miRNA-183 or miRNA-34a (data not shown); dashed horizontal line set to 1.0 for ease of comparison; N = 5; *<i>p</i><0.001 (ANOVA). The results suggest a physiologically relevant miRNA-34a- TREM2-mRNA-3‘UTR interaction and a miRNA-34a-mediated down-regulation of TREM2 expression in stressed MG cells. This pathogenic ineraction may be related to the down-regulation of other immune system genes by up-regulated pro-inflammatory miRNAs in the CNS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref022" target="_blank">22</a>] and/or an impairment in cellular phagocytosis or related phagocytic signaling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref005" target="_blank">5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref007" target="_blank">7</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref020" target="_blank">20</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150211#pone.0150211.ref021" target="_blank">21</a>].</p
Exome Sequencing from Nanogram Amounts of Starting DNA: Comparing Three Approaches
<div><p>Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelect<i><sup>XT2</sup></i> Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.</p></div
Profiles of coverage depth along the target region for Test DNA 1 (upper panel) and Test DNA 2 (lower panel) WES libraries.
<p>Profiles of coverage depth along the target region for Test DNA 1 (upper panel) and Test DNA 2 (lower panel) WES libraries.</p
Sharing of genetic variations between strategies depicted in a Venn diagram.
<p>Only variation with minimum depth of coverage of 20x and minimum quality of 13 were taken into account in all four strategies. The names of the samples are abbreviated: Standard ES = St; ThruPLEX-FD ES = Tp; REPLI-g ES = Rg; GenomePlex ES = Gp. The lower left tile presents the overall statistics, where “Total” indicates the number of all unique SNVs found in the region of interest, i.e. the union of SNV sets found by each strategy.</p
Coverage distribution along the target regions with different percentages of GC bases.
<p>Coverage distribution along the target regions with different percentages of GC bases.</p
Per-base sequencing depth distribution on the target region.
<p>Per-base sequencing depth distribution on the target region.</p
Alignment statistics.
<p>*high confident reads-reads with probability of wrong mapping lower than 0.05 according to their MAPQ score (MAPQ>13).</p><p>**some of GenomePlex ES library reads contained sequences of the primer used for whole genome amplification. These common segments were cut out before the alignment. As a result, 13.8% of and 11.9% of nucleotides were removed from the reads of the Test DNA 1 and Test DNA 2 libraries, respectively.</p><p>***FR-flanking regions (FR), which include 100<b> </b>bp from both ends of the targeted sequences.</p
The experimental scheme.
<p>Two DNA samples (Test DNA 1 and Test DNA 2) were subjected to four exome sequencing (ES) protocols performed in parallel: control (Standard ES) and three modified (REPLI-g ES, GenomePlex ES and ThruPLEX-FD ES). Common steps performed in parallel for several protocols are shown by text boxes spanning the corresponding number of protocol columns.</p
Coverage statistics for the target region.
<p>Analysis was performed on subsets of reads uniquely mapped to the target region and having approximately equal total amounts of bases (<b>∼</b>17<b>×</b>10<sup>8</sup> bases).</p