Arraystar Human Downstream-of-Gene Transcript (DoG RNA) Microarray is designed to profile and study human DoG RNAs. The array includes more than 13,000 probes to simultaneously detect and quantify DoG RNAs, pre-mRNAs of the host genes, and downstream overlapping transcripts as their potential regulatory targets at high accuracy and specificity.

Why Study Downstream-of-Gene Transcripts (DoG RNAs)?

Downstream-of-Gene transcripts (DoG RNAs), a novel type of transcripts derived from protein-coding genes, are generated due to transcription termination failure and read-through transcription.

DoG RNAs modulate gene transcription in diseases>>

• DoG RNAs control the transcription of antisense overlapping genes in cell senescence
• DoG RNAs affect genome 3D organization in viral infection

DoG-derived chimera RNAs and circular RNAs in cancers and diseases>>

DoG RNAs are subject to m6A, R-loop, or epigenetic regulation>>

• Comprehensive coverage of DoG regulatory targets:  
  1) Sense-overlapping lncRNAs, or circRNAs from the DoG downstream neighboring genes
  2) chimera RNAs by cis-splicing of DoG and its downstream neighboring gene transcript
  3) rt-circRNAs by back-splicing of DoG and its read-through downstream neighboring transcript
  4) Anti-sense overlapping mRNAs and lncRNAs
• One array to detect DoGs and all their target RNA types by simple and sensitive array probes specific to the unique splice junctions(Table 1), without the demanding and disparate computational and bioinformatic analyses otherwise required by sequencing method 
• High sensitivity at as low as 1 transcript/cell, good for transcripts even at low abundance (e.g. most of DoG-derived circular RNAs, and chimera RNAs

Table 1. DoG RNA Array probe design method.

Fig.1 Arraystar DoG RNA Microarray probe design.

• DoG Region probe: For DoG detection 3 kb beyond the 3'-terminal polyadenylation signal of the DoG host gene. Not overlapping with the host gene itself.
• Host gene probe: For DoG host gene pre-mRNA detection at its exon-intron junction or within the intron.
• Downstream transcript probe: For detection of DoG downstream mRNA/ lncRNA/ circular RNA/ ChimeraRNA/ rt-circRNA, at its exon-exon junction of the mature linear RNA or back-splicing junction of the mature circular RNA.

Arraystar Human Downstream-of-Gene Transcript (DoG RNA) Array includes more than 13,000 probes to simultaneously detect and quantify DoG RNAs, pre-mRNAs of the host genes, and downstream overlapping transcripts as their potential regulatory targets at high accuracy and specificity.

Table 2. Arraystar Human Downstream-of-Gene Transcript (DoG RNA) Microarray Specifications

Fig. 2. The workflow for Arraystar Downstream-of-Gene Transcript (DoG RNA) Microarray Profiling. The total RNA is primed using a mixture of oligo(dT) and random primers containing T7 polymerase promoter, followed by double-stranded cDNA synthesis by reverse transcriptase. Antisense cRNA is synthesized from the T7 promoter of the double stranded cDNA using in vitro T7 RNA polymerase transcription, simultaneously labeled with Cy3-CTP fluorescent dye along the entire length of the transcript without 3' bias. The purified cRNA is hybridized to Arraystar DoG RNA Array for DoG RNA expression analysis.

Arraystar Downstream-of-Gene Transcript (DoG RNA) Microarray is the most sensitive, effective, and robust method for DoG RNA profiling and analysis. Arraystar has the expertise in downstream-of-gene transcripts and the DoG RNA array profiling technology. Rich analyses and annotation for DoG RNAs are included with the profiling data.

Table 3.  Differentially expressed DoG RNAs, host gene pre-mRNAs and downstream transcripts with systematic lncRNA annotations.

Fig.3. DoG RNA research roadmap

Validation of Differential Expression

The differentially expressed DoG RNAs on the array profiling can be confirmed by independent qPCR quantification of the expression levels. Additionally, RNA fluorescence in situ hybridization (FISH) can provide information about both DoG RNA intensity and DoG RNA subcellular distribution.

Subcellular Fractionation and Localization

DoG RNAs are known to be strongly enriched in the nuclear chromatin fraction, distinguishing them from their cognate mRNAs in the cytoplasm. qPCR analysis of total RNAs isolated from the cytoplasmic, soluble nuclear, and chromatin subcellular fractions can help to understand the DoG RNA subcellular distribution and the functional implications.

CRISPR interference

CRISPR interference (CRISPRi) can be used to knockdown the upstream gene transcription, which helps to investigate its impact on DoG RNA production and the relationship with the DoG RNA. In CRISPRi, catalytically dead Cas9 (dCas9) is guided to the target gene to inhibit its expression without cutting it. dCas9 domain is often fused with a repressive effector (e.g. KRAB domain) to maximize the silencing through steric inhibition of RNA polymerase II (Pol II).

Below is an example using CRISPRi on SRSF1 gene at its promoter, knocking down the expression of both host gene and its DoG RNAs (Fig. 4).

Fig. 4. CRISPRi knockdown of SRSF1 gene transcription (Top) coordinately reduces both the SRSF1 pre-mRNA and DoG RNA to the same ~40% levels as quantified by qPCR (Bottom). Ctrl: Negative control cells without CRISPRi; iS9: Cells with CRISPRi knockdown. e1–i1: SRSF1 pre-mRNA region; DoG1 and DoG2: SRSF1 DoG regions.

Antisense Oligo (ASO)

ASO knockdown of host gene mRNA expression can reduce the levels of the corresponding DoG RNAs. Importantly, ASOs are also known to cause premature transcription termination, which can be particularly useful to target any regions of the DoG host gene to observe how transcription terminations produce DoG RNAs[23, 24]. Transcription termination by ASO (also gapmer here) is mediated by targeted RNA cleavage with endogenous RNase H and requires exonuclease XRN2 similarly to the torpedo model of normal transcription termination[24, 25].

DRIP-seq

R-loops are frequently present at gene promoters and transcription termination regions, both of which play significant roles in the DoG host gene transcription and transcription termination related to DoG RNA formation. DRIP-seq and its variant versions are a common method for R-loop profiling, which can provide valuable insights into R-loop and DoG regulation.

MeRIP-seq

RNA modifications (e.g. m6A) enriched in the transcription termination regions can affect transcription termination. Depletion of the m6A at transcription end sites (TES) results in termination defect and read-through transcription[25]. MeRIP-seq is a tool that can be used to correlate RNA modification with DoG RNAs.

mNET-seq

Mammalian Native Elongating Transcript sequencing (mNET-seq) captures and profiles nascent RNA transcripts still associated with RNA polymerase II, allowing analysis of various stages of transcription, including Pol II pausing, co-transcriptional RNA processing, and termination, related to DoG RNAs.

ChIRP-MS

Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) has been used to identify proteins that interact with RNAs transcriptome wide. By knowing what protein factors are bound to the DoG RNAs, DoG RNA functions and mechanisms can be derived.

Chimera RNA Analysis

Chimera RNA (also known as cis-SAGe) is a class of DoG RNAs formed by cis-splicing of adjacent genes/read-through. siRNA knockdown and qPCR quantification can be used together to dissect chimera RNA expression, function, and molecular mechanism (Fig. 5) [6].

Fig. 5. (A) CTSC-RAB38 chimeraRNA locus depicting the siRNAs targeting CTSC, RAB38, and chimeric CTSC-RAB38 transcripts. These transcripts are quantified by the qPCR primers. The dashed curve represents the splicing that creates the chimeraRNA. (B) Relative CTSC-RAB38 chimeraRNA expression after RNAi knockdowns in CTSC, RAB38, and CTSC-RAB38 regions in three distinct ccRCC cell lines FG2, MF, and ER. *p<0.05 by T-test compared to controls[6].