Epitranscriptomic Array Service

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Arraystar mRNA&lncRNA Epitranscriptomic Microarray and circRNA Epitranscriptomic Microarray quantify your choice of m6A/m5C/m1A/ac4C/m7G/Ψ epitranscriptomic modification in mRNAs & lncRNAs or circRNAs. Please specify one modification per microarray experiment when you request a quote.
For m6A modification, we offer two m6A-RNA enrichment methods to choose from: MeRIP by m6A antibody immunoprecipitation or pull down by m6A reader GST-YTH . 

Benefits

Arraystar Epitranscriptomic Microarrays have unique advantages over MeRIP-seq.
•  A single Epitranscriptomic Microarray to simultaneously profile what gene transcripts are modified, differential modification between conditions, and very importantly, the percentage of modification for each transcript.
•  Excellent coverage for coding and noncoding RNA classes, even for lncRNAs and circRNAs that are difficult to profile by MeRIP-seq.
•  rRNA depletion not required. Faster, simpler than MeRIP-seq.
•  Low sample amount required, starting from as little as 1 ug total RNA for mRNAs & LncRNAs or 3 ug for CircRNAs.

Watch Video> New Discoveries in m6A Epitranscriptomics
Watch Video>  The Latest Highlights on CircRNA in Cancer

Promo: 15% OFF> Valid through 05/31/2024

Service NameSpeciesModificationMethodFormatPrice
mRNA&LncRNA Epitranscriptomic Array Service Human/Mouse/Rat m6A/m5C/m1A/ac4C/m7G/Ψ *Specify one in the quote Antibody 8 x 60K / 4 x 44K
CircRNA Epitranscriptomic Array Service Human/Mouse/Rat m6A/m5C/m1A/ac4C/m7G/Ψ *Specify one in the quote Antibody 8 x 15K
mRNA&LncRNA Epitranscriptomic Array Service Human/Mouse/Rat m6A GST-YTH pull down 8 x 60K / 4 x 44K
CircRNA Epitranscriptomic Array Service Human/Mouse/Rat m6A GST-YTH pull down 8 x 15K

RNA modifications, such as m6A, m1A, m5C, and pseudouridine, together form the epitranscriptome and collectively encode a new layer of gene expression regulation. m6A, the most abundant internal modification in mRNAs and lncRNAs, impacts all aspects of post-transcriptional mRNA/lncRNA metabolism and functions [1]. In addition, m6A are also involved in many other ncRNA functions, including cap-independent translation initiation of circRNA[2], and pri-miRNA processing[3].

The potential effects of RNA modifications depend on not only which gene transcripts, but also the percentage of transcripts that are modified. However, current transcriptome-wide RNA modification profiling methods deal mostly with mapping the modified sites but are unable to quantify the percentage of modified RNA for that transcript. The lack of such quantitative information has been a major concern for scientists (Text box).


The scientists’ top concerns

"The potential effect of an mRNA modification depends on both the molecular consequences and the percentage of transcripts that are modified. For example, a modification that leads to accelerated mRNA decay is unlikely to have much biological effect if only 1% of transcripts are modified, whereas a modification that causes an alternative protein variant to be produced could be functionally important, even at very low levels. A limitation of current m6A and pseudouridine profiling methods is the lack of quantitative information about the extent of modification. Changes in the relative enrichment of a particular sequence in m6A pulldowns from different growth states have been used to infer regulation of modification, but the absolute fraction of mRNA that is modified cannot be determined from these data… High-throughput methods to quantify site-specific m6A and pseudouridine would considerably advance the field.”

[1] Wendy V. Gilbert, Tristan A. Bell, Cassandra Schaening. Science (2016)

"Another important aspect that is yet to be addressed is the dynamics of RNA modification stoichiometry. Current epitranscriptome studies deal mostly with identifying which sites are modified and not the fraction of RNAs in which each site is modified. Low-throughput analysis of m6A modification sites in mRNA and viral RNA shows that no m6A site is modified in 100% of transcripts. Changes in modification stoichiometry may also represent a dynamic parameter of RNA modification biology. As modifications can affect mRNA structure and/or the recruitment of RBPs, modification of a fraction of transcripts at any specific site would generate two distinct mRNA species that differ only in their structures or the readers that bind to them. Therefore, changing modification stoichiometry could represent another mechanism to generate functional diversity from the same RNA transcript. High-throughput methods that can determine modification stoichiometry are needed to address this aspect of the epitranscriptome.”

[4] Cole J.T. Lewis, Tao Pan, Auinash Kalsotra. Nat Rev Mol Cell Biol (2017)


Arraystar Epitranscriptomic Microarrays combine the two-color channel microarray technology with RNA modification immunoprecipitation to quantify the percentage of RNA that are modified at transcript isoform-specific level. The microarrays cover the epitranscriptomes of mRNA, lncRNA, circRNA, pre-miRNA, pri-miRNA, snoRNA, and snRNA classes. The quantitative epitranscriptomic profiling provides the vital information to study the regulatory impacts of RNA modifications.


References

1.  Gilbert WV, Bell TA, Schaening C: Messenger RNA modifications: Form, distribution, and function. Science 2016, 352(6292):1408-1412.[PMID: 27313037]
2.  Yang Y et al: Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Res 2017, 27(5):626-641.[PMID: 28281539]
3.  Alarcon CR et al: HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell 2015, 162(6):1299-1308.[PMID: 26321680]
4.  Lewis CJ, Pan T, Kalsotra A: RNA modifications and structures cooperate to guide RNA-protein interactions. Nat Rev Mol Cell Biol 2017, 18(3):202-210.[PMID: 28144031]
5.  Qin Y et al: TRIM9 short isoform preferentially promotes DNA and RNA virus-induced production of type I interfe*** by recruiting GSK3beta to TBK1. Cell Res 2016, 26(5):613-628.[PMID: 26915459]

GST-YTH is a recombinant fusion protein of YTH-DF2 m6A reader domain (a.a385-579) with a GST tag for m6A RNA enrichment. YTH is an evolutionarily conserved structural domain that selectively "reads" and binds m6A within the consensus RRACH motifs [1]. Structurally, the YTH domain contains two or three tryptophan residues that form an aromatic cage and binding pocket for m6A, with additional interactions with nucleotides before and after m6A, thus giving sequence preference to the RRACH motif [2-4](Fig.1). That is, GST-YTH binds m6A-containing RNAs in an m6A structure- and RRACH sequence motif-dependent manner [1], both of which are distinct from m6Am and other similar RNA modifications. Thus, GST-YTH pulldown is highly specific to m6A without the cross-reactivity with other structurally similar RNA modifications[5], particularly m6Am [6], as by m6A antibody MeRIP.

YTH-1

Figure 1. YTH binds to m6A-modified RNAs in an m6A structure- and RRACH sequence motif-dependent manner, at higher specificity to m6A without the cross-reactivity to other similar RNA modifications such as m6Am.


Reference
1.  Wang X et al: N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 2014, 505(7481):117-120.[PMID: 24284625]
2.  Luo S, Tong L: Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain. Proc Natl Acad Sci U S A 2014, 111(38):13834-13839.[PMID: 25201973]
3.  Theler D et al: Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA. Nucleic Acids Res 2014, 42(22):13911-13919.[PMID: 25389274]
4.  Xu C et al: Structural basis for selective binding of m6A RNA by the YTHDC1 YTH domain. Nat Chem Biol 2014, 10(11):927-929.[PMID: 25242552]
5.  Linder B et al: Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 2015, 12(8):767-772.[PMID: 26121403]
6.  https://sysy.com/product/202003

Quantifying the percentage of modification for each transcript by microarray

The modified and unmodified fractions of the same RNA transcript, which differ only in their structures or the readers that bind to them, can assume different fates [4] (Figure 1). Importantly, the percentage of modified transcripts is highly relevant to their functional consequences. While current RNA modification profiling methods, such as MeRIP-seq (i.e. m6A-seq), can map the modification locations, they do not quantify the relative fraction of modified and unmodified RNA for a given transcript. Arraystar Epitranscriptomic Microarrays have the power to determine the percentage of modified transcripts by measuring the percentage of modification for each transcript in two color channels on the same array (Figure 2), while simultaneously profile what gene transcripts are modified and the differential modification between conditions.

The changing modification stoichiometry generates functional diversity from the same RNA transcript

Fig 1. The changing modification stoichiometry generates functional diversity from the same RNA transcript. The percentage of modified RNA “transcript a” can be very low under one cellular condition (e.g. Cell state 1), but change to high (e.g. Cell state 2) under another cellular condition. By causing RNA structural changes and direct recruitment of modification reader proteins, the modified “transcript a” acquires a different fate, for example, from protein translation to increased RNA decay.

Arraystar Epitranscriptomic Microarray

Fig 2. Arraystar Epitranscriptomic Microarray detects the immunoprecipitated modified RNA in Cy5 and supernatant unmodified RNA in Cy3 channels on the same array, such that the modified and unmodified percentages for each transcript can be measured. Alternatively spliced transcript isoforms are specifically and unambiguously detected by transcript-specific array probes.
 

Coverage of coding and noncoding epitranscriptomes

•   Arraystar mRNA&lncRNA Epitranscriptomic Microarrays
For mRNA, lncRNA, and mid-sized noncoding RNA classes of pre-miRNA, pri-miRNA, snoRNA, and snRNA.
•   Arraystar circRNA Epitranscriptomic Microarrays
For circular RNAs, at high confidence collection of observed expression in  >= 2 experiments and  >= 4 samples.
•   High sensitivity and accuracy, even for RNAs difficult for MeRIP-seq (e.g. lncRNAs and circRNAs).
As a rule of thumb, the abundance of LncRNAs/circRNA junction sites in samples is too low to be accurate quantified by sequencing, and similarly for the abundance of IP-enriched LncRNAs/circRNA junctions. 
What Are the Limitations of RNA-seq for LncRNA Profiling? >>
Why Use Microarray Over RNA-seq for Circular RNA Expression Profiling? >>
 

RNA modifications at transcript-specific level

Alternatively spliced transcript isoforms have tissue-specific expression and distinct biological functions. For example, TRIM9 short isoform (NM_052978), not the long isoform (NM_015163), selectively inhibits the production of pro-inflammatory cytokines in response to viral infection[5]. Changes in their percentages of modified transcript isoforms have been associated with biological functions and diseases.

Arraystar Epitranscriptomic Microarrays use specific exon or splice junction probes to reliably and accurately profile the RNA modification in each individual transcript isoform, defining a new level of details of epitranscriptomics (Fig. 3).

Arraystar transcript-specific Probe

Fig 3.  Arraystar Epitranscriptomic Microarray uses transcript-specific Probe A and Probe B to distinctly detect RNA modification in TRIM9 long (NM_015163) and short (NM_052978) transcript isoforms.
 

Low sample amount requirements

Current MeRIP-seq technique requires a massive amount of total RNA at  >= 120 µg as the starting material, making it difficult to study on samples of limited availability. Arraystar Epitranscriptomic Microarray requires as little as is 3 µg total RNA, which is magnitudes lower than MeRIP-seq. The low sample amount requirement opens up opportunities for research projects where the samples are rare or of limited supply.
 

Human mRNA&lncRNA Epitranscriptomic microarray

Total number of distinct probes 60,613
Probe length 60 nt
Probe site mRNA and lncRNA: Specific exon or splice junction sequence along the entire length of transcript.

Mid-size ncRNAs: Unique sequence regions in ncRNA

Probe specificity Transcript-specific
Labeling method cRNAs are labeled along the entire length without 3’ bias, even for degraded RNA at low abundance
Protein coding mRNAs 44,122
LncRNAs 12,496
Mid-size ncRNAs 1,366 (pre-miRNAs), 1,642 (pri-miRNAs), 19 (snRNAs), 786 (snoRNAs)
mRNA sources Refseq, UCSC, GENECODE, FANTOM5 CAT [2-6]
LncRNA sources Arraystar LncRNA collection pipelines:

lncRNAs from all databases and literatures up to 2018. “Canonical” or “longest” priority assigned to transcript for each lncRNA gene.

External Databases (current in 2018):
Refseq, UCSC, GENCODE, FANTOM5 CAT, LncRNAdb, RNAdb, NRED [2-8]

Literatures:
Scientific publications up to 2018 [9-45]

Mid-size ncRNA sources GENECODE, miRBase [1-2]
Array Format 8 × 60 K

Reference List


Mouse mRNA&lncRNA Epitranscriptomic microarray

Total number of distinct probes

60,773

Probe length 60nt
Probe site LncRNA and mRNA: Specific exon or splice junction sequence along the entire length of transcript

Mid-size ncRNAs: unique sequence regions in ncRNAs

Probe specificity Transcripts specific
Labeling method cRNAs are labeled along the entire length without 3’ bias, even for degraded RNA at low abundance
Protein coding mRNAs 48,161
LncRNAs 8,393
Mid-size ncRNAs 701 (pre-miRNAs), 957 (pri-miRNAs), 1229 (snRNAs), 1,200 (snoRNAs)
LncRNA sources Arraystar LncRNA collection pipelines:

lncRNAs from all databases and literatures up to 2018. “Canonical” or “longest” priority assigned to transcript of each lncRNA gene.

External Databases:
Refseq, UCSC, GENCODE, GenBank, lncRNAdb, NRED [2-7]

Literatures:
lincRNA catalogs [8-11], T-UCRs [12], Evolutionary constrained LncRNAs [13], lncRNA publications up to 2018 [14-22]

mRNA sources Refseq, UCSC, GENECODE [2-3]
Mid-size ncRNA sources GENECODE, miRBase [1-2]
Array Format 8 × 60 K

Reference List


Rat mRNA&lncRNA Epitranscriptomic microarray

Total number of distinct probes 40,991
Probe length 60nt
Probe site LncRNA and mRNA: Specific exon or splice junction sequence along the entire length of transcript

Mid-size ncRNAs: specific sequence regions in ncRNAs

Probe specificity Transcripts-specific
Labeling method cRNAs are labeled along the entire length without 3’ bias, even for degraded RNA at low abundance
Protein coding mRNAs 27,770
LncRNAs 10,582
Mid-size ncRNAs 432 (pre-miRNAs), 449 (pri-miRNAs), 155 (snRNAs), 1,469 (snoRNAs)
LncRNA sources Arraystar LncRNA collection pipelines:

lncRNAs from databases and scientific publications up to 2018. “Canonical” or “longest” priority assigned to transcript for each lncRNA gene

Updated Databases:
Ensembl 92 [1] , Refseq [5]

Literatures:
T-UCRs [6-9]

mRNA sources Refseq, Ensembl 92[1, 5]
Mid-size ncRNA sources GENECODE, miRBase, GtRNADb [2-3]
Array Format 4 × 44K

Reference List


Human circRNA Epitranscriptomic Microarray 

Total number of distinct probes 13,617
Probe length 60nt
Probe site circular junctions
Probe specificity Transcript-specific
Labeling method Random primer labeling coupled with RNase R sample pretreatment to ensure specific and efficient labeling of circular RNAs
circRNAs 13,617
circRNA sources Updated Databases: circbase, CircNet, circRNADb [1-3]

Literatures: [4-10]

Array Format 8 × 15 K

Reference List


Mouse circRNA Epitranscriptomic Microarray

Total number of distinct probes 14,236
Probe length 60nt
Probe site Circular junctions
Probe specificity Transcript-specific
Labeling method Random primer labeling coupled with RNase R sample pretreatment to ensure specific and efficient labeling of circular RNAs
circRNAs 14,236
circRNA sources Updated Databases: circbase, CircNet, circRNADb [1-3]

Literatures: scientific publications [4-5]

Array Format 8 × 15 K

Reference List


Rat circRNA Epitranscriptomic Microarray

Total number of distinct probes 14,145
Probe length 60nt
Probe site Circular junctions
Probe specificity Transcript-specific
Labeling method Random primer labeling coupled with RNase R sample pretreatment to ensure specific and efficient labeling of circular RNAs
circRNAs 14,145
circRNA sources Updated Databases: circbase, CircNet, circRNADb [1-3]

Literatures: scientific publications [4-5]

Array Format 8 × 15 K

Reference List

Arraystar Epitranscriptomic Microarray profiling is provided as a full service, from sample preparation, RNA immunoprecipitation/GST-YTH pull down, cRNA labeling, microarray experiment, to annotation and data analysis. The in-process QC steps are included to ensure data quality and success. Just send us your samples, and we'll do the rest!

mRNA&lncRNA Epitranscriptomic Microarray Workflow

Take m6A modification as an example:

YTH-2

Fig 1. mRNA&lncRNA Epitranscriptomic Microarray Workflow.
       •  RNA samples provided by Customer (See Sample Submission for details)
       •  RNA QC
       •  m6A-enrichment by m6A-MeRIP or GST-YTH pulldown
       •  cRNA synthesis and two-color labeling (Cy5 for enriched-RNA and Cy3 for supernatant RNA)
       •  Array hybridization, washing, and scanning
       •  Data extraction, annotation, analysis and summarization

 

circRNA Epitranscriptomic Microarray Workflow

Take m6A modification as an example:

YTH-3

Fig 2. circRNA Epitranscriptomic Microarray Workflow.
       •  RNA samples provided by Customer (See Sample Submission for details)
       •  RNA QC
       •  m6A-enrichment by m6A-MeRIP or GST-YTH pulldown
       •  RNase R treatment to remove linear RNAs (e.g. rRNA, lncRNA, mRNA, etc)
       •  cRNA synthesis and labeling (Cy5 for enriched-RNA and Cy3 for supernatant RNA)
       •  Array hybridization, washing, and scanning
       •  Data extraction, annotation, analysis and summarization

Arraystar has the expertise and in-depth knowledge in microarray profiling, data analysis, and result interpretation. Rich and detailed epitranscriptomic bioinformatic analyses are provided in the data files and included in the service.

Differentially modified transcripts (mRNAs, lncRNAs, and mid-size ncRNAs)

epitranscriptomic

Differentially modified circRNAs

epi-circ-1

epi-circ-2


Hierarchical clustering heatmap of differentially modified RNAs

6


GO Enrichment Analysis of differentially modified mRNAs

GO-1


Pathway analysis of differentially modified mRNAs

pathway-2

CircRNA Epitranscriptomic Array Service

N6-methyladenosine hypomethylation of circGPATCH2L regulates DNA damage and apoptosis through TRIM28 in intervertebral disc degeneration. Chen Z, et al. Cell Death & Differentiation, 2023

YTHDC1 maintains trophoblasts function by promoting degradation of mA6-modified circMPP1. Wang D, et al. Biochemical Pharmacology, 2023

METTL14-mediated m6A modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis. Fan H N, et al. Molecular Cancer, 2022

The Combined Effects of circRNA Methylation Promote Pulmonary Fibrosis. Wang S, et al. American Journal of Respiratory Cell and Molecular Biology, 2022

m6 A-modified circFOXK2 targets GLUT1 to accelerate oral squamous cell carcinoma aerobic glycolysis. Cui Y,et al. Cancer Gene Therapy, 2022

High-throughput microarray reveals the epitranscriptome-wide landscape of m6A-modified circRNA in oral squamous cell carcinoma. Zhao W,et al. BMC genomics, 2022

Chronic Hexavalent Chromium Exposure Up-regulates the RNA Methyltransferase METTL3 Expression to Promote Cell Transformation, Cancer Stem Cell-like Property and Tumorigenesis. Wang Z, et al. Toxicological Sciences, 2022

More Publications>>

 

mRNA&LncRNA Epitranscriptomic Array Service

Multigenerational paternal obesity enhances the susceptibility to male subfertility in offspring via Wt1 N6-methyladenosine modification. Xiong Y W, et al, Nature Communications, 2024

RNA N6-methyladenosine modification based biomarkers for absorbed ionizing radiation dose estimation. Chen H, et al. Nature Communications, 2023

Sperm Rhoa m6A modification mediates intergenerational transmission of paternally acquired hippocampal neuronal senescence and cognitive deficits after combined exposure to environmental cadmium and high-fat diet in mice. Zhang J, et al. Journal of Hazardous Materials, 2023

N6 -methyladenosine promotes aberrant redox homeostasis required for arsenic carcinogenesis by controlling the adaptation of key antioxidant enzymes. Zhao T, et al. Journal of Hazardous Materials, 2023

N6-methyladenosine demethylase FTO regulates synaptic and cognitive impairment by destabilizing PTEN mRNA in hypoxic ischemic neonatal rats. Deng J, et al. Cell Death & Disease, 2023

High glucose induces tau hyperphosphorylation in hippocampal neurons via inhibition of ALKBH5-mediated Dgkh m6 A demethylation: a potential mechanism for diabetic cognitive dysfunction. Qu M, et al. Cell Death & Disease, 2023

METTL3/IGF2BP3 axis inhibits tumor immune surveillance by upregulating N6-methyladenosine modification of PD-L1 mRNA in breast cancer. Wan W, et al. Molecular Cancer, 2022

N6 -methyladenosine plays a dual role in arsenic carcinogenesis by temporal-specific control of core target AKT1. Zhao T,et al. Journal of Hazardous Materials, 2022

WTAP-mediated m6A modification of lncRNA DIAPH1-AS1 enhances its stability to facilitate nasopharyngeal carcinoma growth and metastasis. Li Z X, et al. Cell Death & Differentiation, 2022

N6 -methyladenosine plays a dual role in arsenic carcinogenesis by temporal-specific control of core target AKT1. Zhao T,et al. Journal of Hazardous Materials, 2022

PGE2-EP3 axis promotes brown adipose tissue formation through stabilization of WTAP RNA methyltransferase. Tao X, et al. The EMBO Journal, 2022

METTL3 promotes colorectal carcinoma progression by regulating the m6A–CRB3–Hippo axis. Pan J, et al. Journal of Experimental & Clinical Cancer Research, 2022

ALKBH5 regulates cardiomyocyte proliferation and heart regeneration by demethylating the mRNA of YTHDF1. Han Z, et al. Theranostics, 2021

RBM15 facilitates laryngeal squamous cell carcinoma progression by regulating TMBIM6 stability through IGF2BP3 dependent. Wang X, et al. Journal of Experimental & Clinical Cancer Research, 2021

ALKBH5-mediated m6A mRNA Methylation Governs Human Embryonic Stem Cell Cardiac Commitment. Han Z, et al. Molecular Therapy-Nucleic Acids, 2021

More Publications>>