taqman probes snp microarray sequencing of donor dna  (Thermo Fisher)

Journal: Nature Communications

Article Title: Two nuclear effectors of the rice blast fungus modulate host immunity via transcriptional reprogramming

doi: 10.1038/s41467-020-19624-w

Figure Lengend Snippet: a Effector binding elements (EBEs) targeted by MoHTR1 and MoHTR2 were identified using a protein binding microarray. Each MoHTR-Δsp:DsRed (R) protein produced using E . coli was applied to DNA chip containing quadrupled 9-mer nucleotides. The EBE for each protein was identified based on the fluorescent intensity from the group of elements containing the same 8-mer nucleotide. b Effect of a single nucleotide mismatch in each EBE on the binding affinity to MoHTR. The line denoted using + indicates the signal intensity from EBE without variation. Bars represent the average signal intensity from individual EBE variants with each carrying a different single-nucleotide mismatch (noted in x -axis). c Schematic representation of a yeast one-hybrid assay used for detecting MoHTR–EBE interaction and resulting data. NLS:AD:MoHTR-Δsp was expressed under the ADH1 promoter. NLS, a nuclear localization signal from the Simian Virus 40 large T antigen; AD, GAL4 activation domain. The reporter, β-galactosidase (GAL), was placed under the recombinant promoter comprised of an EBE-centered 38-bp fragment and a minimal cytochrome C1 promoter (CYC1). GAL expression was activated when NLS:AD:MoHTR-Δsp binds to its EBE in the promoter. pLacZi was used as a negative control. d Validation for binding of MoHTRs and target promoters in planta. LUC was regulated by the corresponding gene promoter. Activation domain of TAL effector (AD) was linked to MoHTR -Δsp and expressed under the 35S CaMV promoter. e Relative LUC activities in rice protoplasts transfected with AD-linked MoHTR -Δsp were compared with those transfected with the EV and corresponding reporter vector (M4: OsMYB4 promoter, W45: OsWRKY45 promoter). n = 3 independently transfected protoplasts; mean ± standard deviation (SD); * P < 0.01, two-sided Student’s t -tests. Source data underlying Fig. 3e are provided as a Source Data file.

Article Snippet: The DNA microarray used contains 232,145 probes that consist of quadruple 9-mer oligonucleotides and cover all possible 9-mer sequences (Agilent Technology).

Techniques: Binding Assay, Protein Binding, Microarray, Produced, Y1H Assay, Activation Assay, Recombinant, Expressing, Negative Control, Transfection, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Neuroscience

Article Title: A Neuronal Transcriptome Response Involving Stress Pathways is Buffered by Neuronal microRNAs

doi: 10.3389/fnins.2012.00156

Figure Lengend Snippet: Stress induces expression of genes subject to miRNA-mediated regulation. (A) The x -axis shows differential expression (log 2 ) between mock transfected and untransfected cultures, the y -axis shows the adjusted P -value ( − log 10 ) for differential expression. Each point on the plot uniquely represents one gene, genes above the dashed line are differentially expressed with multiple testing adjusted P < 0.1. The insets show enrichment (log 2 ) of genes either downregulated (white bars) or induced (black bars) by stressful treatments. Genes differentially expressed upon KCl treatment where identified by Illumina microarrays (Materials and Methods). Genes differentially expressed upon kainite treatment where derived from published microarray profiling data (Akahoshi et al., ; Materials and Methods). Mouse homologs (from HomoloGene; Sayers et al., ) of human genes that were previously reported as differentially expressed upon aging of the human brain comprised the aging sets. Double asterisks indicate hypergeometric P < 0.001 (Materials and Methods). The set of 10,849 genes detected by Illumina microarrays (Materials and Methods) in mock transfected and untransfected cultures was used as the universe for the hypergeometric tests. (B) The x -axis represents 3′UTRs corresponding to expressed genes sorted from most downregulated to most upregulated in comparison of mock transfected vs. untransfected cultures. P -values are calculated using the Sylamer method (van Dongen et al., ). Positive values on the y -axes represent nucleotide word enrichment [+|log 10 ( P -value)|] and negative values represent depletion [−|log 10 ( P -value)|]. The red and blue lines show enrichment profiles of 7(2) or 7(1A)-type seed matching sites (Bartel, ) for miR-124, the gray lines – for other miRNAs (Materials and Methods).

Article Snippet: Illumina microarray probe sequences were taken from the Illumina mRNA array annotation file MouseWG-6_V2_0_R2_11278593_A (BGX file) available from the Illumina website .

Techniques: Expressing, Quantitative Proteomics, Transfection, Derivative Assay, Microarray, Comparison

Journal: Frontiers in Neuroscience

Article Title: A Neuronal Transcriptome Response Involving Stress Pathways is Buffered by Neuronal microRNAs

doi: 10.3389/fnins.2012.00156

Figure Lengend Snippet: Comparison with previously published miR-124 targets . (A,B) The Venn diagrams show counts of putative direct targets of miR-124 that were inferred from the transfection experiments in this work and from the published HITS-CLIP sequencing (Chi et al., ) and microarray (Lim et al., ) experiments. The latter study is based on HeLa transfrection experiment. The mouse homologs of human miR-124 targets were retrieved from HomoloGene http://www.ncbi.nlm.nih.gov/homologene ; (C) the Venn diagram shows counts of miR-124 targets reported in HeLa study (Lim et al., ) and transcripts induced by transfection alone in our experiment. In all cases the test universe was 3,465 mouse transcripts, with 3′UTRs containing one or more 7(2) or 7(1A)-type seed matching site for miR-124. The test universe was a complete list of mouse genes encoding transcripts with 3′UTRs containing one or more 7(2) or 7(1A)-type seed matching site for miR-124, and which were represented on Illumina Sentrix BeadChip Array Mouse-WG6 v2 microarray platform (3,465 genes in total). The text shows fold enrichment above what is expected by chance alone and the hypergeometric P -value for the intersection.

Article Snippet: Illumina microarray probe sequences were taken from the Illumina mRNA array annotation file MouseWG-6_V2_0_R2_11278593_A (BGX file) available from the Illumina website .

Techniques: Comparison, Transfection, Sequencing, Microarray