2x oratm see qpcr probe mix  (highQu Inc)

Journal: Nucleic Acids Research

Article Title: SETDB1 activity is globally directed by H3K14 acetylation via its Triple Tudor Domain

doi: 10.1093/nar/gkae1053

Figure Lengend Snippet: SETDB1 is recruited to H3K14ac containing target loci. ( A ) Scheme of the cell HCT116 derived lines generated in this study by SETDB1 KO followed by reconstitution with SETDB1 WT or mutants and by HBO1 KO. ( B and C ) H3K9me3 (B) and H3K14ac (C) ChIP-qPCR at known SETDB1 target regions ( , ) showing changes in the histone modification as indicated upon SETDB1 KO and reconstitution with WT SETDB1 of its catalytically inactive mutant H1224K or the 3TD mutant F332A. Moreover, HBO1 KO cells were investigated. ChIP was performed on mononucleosomes isolated from two individual biological replicates from each cell line (represented as dots). Averages of the individual measurements are represented as bars.

Article Snippet: For each region of interest, a master mix was prepared with 7.5 μl of 2X ORATM See qPCR Probe Mix (highQu), 0.4 μl forward primer, 0.4 μl reverse primer and 5.7 μl ddH 2 O.

Techniques: Derivative Assay, Generated, ChIP-qPCR, Modification, Mutagenesis, Isolation

Journal: Journal of Molecular Cell Biology

Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress

doi: 10.1093/jmcb/mjz094

Figure Lengend Snippet: Hypoxia-induced changes in circRNA levels. ( A ) Sixty-four circRNAs significantly change in abundance upon hypoxia. Volcano plots show log 2 -transformed moderated fold changes in expression (hypoxia over normoxia, taken from DESeq2) of circRNAs in the three cancer cell lines against associated P -values (−log 10 ). Differentially expressed circRNAs are highlighted in red (FDR < 0.05). Labels indicate circRNAs that were validated by RT-qPCR in E and F . Only high-confidence circRNAs with at least 5 reads in any 2 samples of a single cell line were tested for differential expression. ( B ) circPLOD2 is consistently upregulated upon hypoxia. Genome browser view of exons 2–3 of the PLOD2 gene. The gene is located on the minus strand. Chimeric alignments (back-splice reads) from RNA-Seq data for MCF-7 and HeLa cells under normoxic and hypoxic conditions are shown. ( C ) Many upregulated circRNAs originate from upregulated host genes. Scatterplot compares log 2 -transformed moderated fold changes in expression (hypoxia over normoxia, taken from DESeq2) of 64 hypoxia-regulated circRNAs and their host genes in the three cancer cell lines (indicated by color). Pearson correlation coefficients and associated P -values are given for each cell line. Selected circRNAs are labeled. ( D ) Most circRNAs do not change in back-splicing rate between conditions. Scatterplots compare circular-to-linear ratios (CLRs) of all high-confidence circRNAs in the three cell lines under hypoxic and normoxic conditions. Red lines indicate >2-fold change in CLR between conditions. The hypoxia-regulated circRNAs (significantly changed in overall abundance according to DESeq2) in each cell line (as shown in A ) are highlighted in orange. ( E and F ) Expression changes of hypoxia-regulated (bold) and control (regular) circRNAs in HeLa ( E ) and MCF-7 ( F ) cells kept in hypoxic conditions (24 h and 48 h, respectively). Dot plot shows relative circRNA levels (over normoxia) based on RT-qPCR normalized to U6 snRNA for HeLa and P0 ( MPZ ) for MCF-7 . Mean and standard deviation of the mean are shown together with red circles for replicate measurements. In HeLa cells, all seven expected circRNAs were significantly upregulated ( n = 3, * P < 0.05, ** P < 0.01). We additionally included intronic circZNF292, known to be hypoxia-induced in HUVEC cells , although its regulation did not reach significance in our RNA-Seq data analysis. In MCF-7 cells, six of the seven expected circRNAs were significantly upregulated ( n ≥ 3, * P < 0.05, ** P < 0.01), as well as circHIPK3 that was not found as significantly regulated in the RNA-Seq data. Upregulation of VEGFA mRNA served as control for hypoxia treatment.

Article Snippet: Differential expression was validated by RT-qPCR using 1× final concentration of 2X ORA qPCR Green ROX L Mix (highQu GmbH), 500–2000 nM forward and reverse primers (depending on the primer) and 1 μl of 1:8 dilution of cDNA.

Techniques: Transformation Assay, Expressing, Quantitative RT-PCR, Quantitative Proteomics, RNA Sequencing, Labeling, Control, Standard Deviation

Journal: Journal of Molecular Cell Biology

Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress

doi: 10.1093/jmcb/mjz094

Figure Lengend Snippet: Biogenesis of hypoxia-regulated circRNAs. ( A ) Schematic visualization of how pairing of inverted Alu elements in flanking introns can promote circRNA formation. ( B ) circRNA-flanking introns are enriched in Alu elements. Barchart shows the percentage of introns with Alu elements in a 500-bp window next to splice sites of annotated internal exons (Ctrl, white) compared to back-splice sites of all (All, dark gray) and hypoxia-regulated (Reg, light gray) circRNAs. ( C ) circRNA-flanking Alu element pairs are more often in inverted orientation. Barchart shows the percentage of Alu element pairs in the same (gray) or inverted (blue) orientation. Exon categories and windows as in B . ( D ) Some regulated circRNAs harbor extended complementary sequences in their flanking introns. Barchart depicts that length and identity of longest local alignment (left and right scales, respectively) from pairwise alignments of flanking introns (500-bp window) for the 64 hypoxia-regulated circRNAs. The upper and lower dashed lines indicate 85% identity and a minimum alignment length of 40 nt, respectively. Mutation experiments demonstrated that inverted repeats of 30–40 nt are sufficient to promote circularization . ( E ) HNRNPC binding shows more binding at back-splice sites compared to linearly spliced exons. Metaprofile of HNRNPC binding (iCLIP) in a 300-nt window around back-splice sites, including 250 nt intron and 50 nt into the circularized exons. circRNAs of the high-confidence set expressed in HeLa ( n = 1133) were separated into hypoxia-regulated and non-regulated circRNAs and compared to 4853 linear exons from expressed PCGs that do not undergo circularization. For each position, the mean coverage in each set is shown by a dot. Lines were smoothed with locally weighted polynomial regression (loess, span = 0.05). ( F ) HNRNPC binds upstream of the 3′ back-splice site of circSMARCA5, which is upregulated upon HNRNPC depletion. Genome browser view shows section of the SMARCA5 gene including RNA-Seq data (chimeric alignments) for HeLa cells under normoxic conditions (top) and HNRNPC iCLIP data from HeLa cells. Binding sites predicted by PureCLIP are shown below. ( G ) Expression changes of a panel of 25 circRNAs upon HNRNPC depletion with two independent siRNAs in HeLa cells. Barchart shows the relative circRNA levels based on RT-qPCR normalized to U6 snRNA. Data were shown as mean ± SD. circCDYL2, circRARS, and circSMARCA5 were significantly deregulated ( n = 3, * P < 0.05, ** P < 0.01).

Article Snippet: Differential expression was validated by RT-qPCR using 1× final concentration of 2X ORA qPCR Green ROX L Mix (highQu GmbH), 500–2000 nM forward and reverse primers (depending on the primer) and 1 μl of 1:8 dilution of cDNA.

Techniques: Mutagenesis, Binding Assay, RNA Sequencing, Expressing, Quantitative RT-PCR