Epitranscriptomic Array Service

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Arraystar m6A-mRNA&lncRNA Epitranscriptomic Microarray Service and m6A-circRNA Epitranscriptomic Microarray Service quantify the most prominent m6A epitranscriptomic modification in mRNAs & lncRNAs or circRNAs, respectively.

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 µg total RNA.
•  Suitable for more sample types, such as degraded FFPE, and serum/plasma/whole blood samples.

Service NameModificationDescriptionFormatPrice
Human mRNA&lncRNA Epitranscriptomic Array Service m6A 44,122mRNAs; 12,496lncRNAs; 3,813Mid-size ncRNAs 8*60K
Mouse mRNA&lncRNA Epitranscriptomic Array Service m6A 48,161mRNAs; 8,393lncRNAs; 4,087Mid-size ncRNAs 8*60K
Rat mRNA&lncRNA Epitranscriptomic Array Service m6A 27,770mRNAs; 10,582lncRNAs; 2,505Mid-size ncRNAs 4*44K
Human circRNA Epitranscriptomic Array Service m6A 13,617 circular RNAs 8*15K
Mouse circRNA Epitranscriptomic Array Service m6A 14,236 circular RNAs 8*15K
Rat circRNA Epitranscriptomic Array Service m6A 14,145 circular RNAs 8*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 interferon by recruiting GSK3beta to TBK1. Cell Res 2016, 26(5):613-628.[PMID: 26915459]

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.

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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.

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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).

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), preferentially promotes DNA and RNA virus-induced production of type I interferon [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).

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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 1 µg total RNA, which is magnitudes lower than MeRIP-seq (Table 1). The low sample amount requirement opens up opportunities for research projects where the samples are rare or of limited supply.

Table 1. Sample requirements for Arraystar Epitranscriptomic Microarray and MeRIP-seq.

Sample requirements Epitranscriptomic Microarray MeRIP-seq
Starting material amount >=1ug total RNA >=120ug total RNA
mRNA isolation or rRNA removal Not required Required
Intact RNA Not required Required


 

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, MeRIP, 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!

m6A-mRNA&lncRNA Epitranscriptomic Microarray workflow

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

 

m6A-circRNA Epitranscriptomic Microarray

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Fig 2. m6A-circRNA Epitranscriptomic Microarray Workflow.
       •  RNA samples provided by Customer (See Sample Submission for details)
       •  RNA QC
       •  m6A-RIP
       •  RNase R treatment to remove linear RNAs (e.g. rRNA, lncRNA, mRNA, etc)
       •  cRNA synthesis and labeling (Cy5 for IP-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 m6A-methylated transcripts (mRNAs, lncRNAs, and mid-size ncRNAs)

Epi-lncRNA_and_mRNA


Differentially m6A-methylated circRNAs

epi-circRNA

Hierarchical clustering heatmap of differentially m6A-methylated RNAs

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GO Enrichment Analysis of differentially m6A-methylated mRNAs

GO-1


Pathway analysis of differentially m6A-methylated mRNAs

pathway-2