Tiling probes for 481 UCRs and their extended regions
481 UCR DNA segments are extended for 1 kb flanking each end of the UCR. Each extended UCR is tiled by sense and antisense strand-specific probes at 40 bp resolution. A total of 51,320 tiling probes, covering 481 UCRs and their extended regions, are printed onto the Arraystar Human T-UCR microarray.
Figure 1. Tiling probes of the Arraystar Human T-UCR Microarray. UCRs, plus an additional 1 kb of genomic sequence flanking each end, are represented by both sense (red arrows) and antisense (purple arrows) tiling probes at 40 bp resolution. Because the probe length is 60 nt and the probe spacing is set to 40 nt, probes overlaps each other by 20 nucleotides.
Transcript-specific probes for potential T-UCRs and RNA transcripts overlapping UCR Loci
153 potential T-UCRs and 3,789 RNA transcripts (LncRNAs and mRNAs) overlapping UCR loci are collected from the most authoritative databases, such as RefSeq, UCSC known genes, Ensembl, and GenBank. Multiple RNA transcripts occurring in the same transcriptional orientation at one UCR locus are considered to be different isoforms of the same gene. Each transcript is detected accurately using one specific exon or splice junction probe. The strategy to design transcript-specific probes is as follows: First, genome sequences corresponding to each transcript are divided into small elements ("exon" elements and "splice-junction" elements) based on the coordinates of these transcripts; second, one probe is designed for each element; and finally, probes specific for each transcript and probes specific for each gene (one probe common to all the transcripts in the gene locus) are identified. Using this method, a total of 6,438 probes were designed to detect the expression of 3,789 individual RNA transcripts.
Figure 2. Design method for transcript-specific probes. Two putative transcripts (transcripts A and B of gene I) overlapping one UCR locus are aligned to the genome. 4 exon elements (F1, F2, F3 and F4) and 3 splice junction elements (J1, J2 and J3) are identified. The probes designed to detect splice junction elements J2 and J3, and exon element F3, are probes specific for transcripts A or B. Probes obtained from exon elements F1, F2 and F4, and from splice element J1, are used as gene I-specific probes.
Gene-specific probes for RefSeq genes proximal to UCRs
1,809 coding genes located within 500 kb of UCRs are detected by gene-specific probes. Generally, 1 gene-specific probe is designed for each gene locus.
Probe design example of Arraystar human T-UCR Microarray
Figure 3. UCR probe design strategy. The UCR uc.144 is given here as an example. uc.144 plus an additional 1kb of genomic sequence flanking each end is tiled by strand-specific probes (forward and reverse probes are represented by red and violet vertical bars, respectively). In addition, by using transcript-specific probes [exon probe (yellow bar) or specific junction probe (yellow line)], two RefSeq transcripts (NM_031372 and NR_003249) overlapping uc.144 can be specifically detected. Further, the proximal gene HNRNPD, located within 500 kb of uc.144, is represented by a gene specific probe (black bar), to examine any possible coordinated expression between the T-UCR of uc.144 and HNRNPD.