N6-methyladenosine is the most abundant RNA modification in mammalian mRNA and long non-coding RNA, occurring on average in three to five sites per transcript[1,2]. Profiling m6A at single nucleotide resolution has been challenging. m6A on lower abundance mRNAs/lncRNAs, the inert reactivity of the methyl group, and interference from RNA structure near the modification site further contribute to the difficulties^[1]. The widely used m6A/MeRIP-seq based methods use m6A-antibody to immunoprecipitate m6A-modified RNA fragments and RNA-seq to locate the m6A sites within 200-nt ^{[3, 4]}. Although these approaches have helped the analysis of m6A epitranscriptomics possible^[5-9], they cannot precisely identify which adenosines in a MeRIP-seq peak are actually modified, nor can they quantify the modification fraction for each site^[1]. New methods that can unambiguously and quantitatively determine the m6A status and the percentage of modification at single nucleotide resolution are urgently needed to further advance the understanding of the molecular and the biological functions of m6A epitranscriptomics. To address these challenges, Arraystar have developed Arraystar m6A single nucleotide arrays that precisely locate the exact m6A modification at single-nucleotide resolution and quantify the stoichiometry of m6A modification fractions.

•  Limited specificity and sensitivity

Due to the m6A-antibody cross-reactivity with other related modifications (e.g. m6Am)^{[4, 5, 11, 12]}, the ​assay specificity ​​to m6A modification is limited. Also, in the absence of an ​orthogonal technique as an independent reference, the ​sensitivity of m6A-antibody based m6A profiling has not been systematically evaluated. The microarrays are thus an antibody-independent method for this critical need^[5].

Solution: For the first time, the microarrays based on methyl-sensitive MazF RNase now allow systematic m6A profiling independent of m6A-antibody immunoprecipitation based approaches such as MeRIP or miCLIP.

•  Limited ​resolution

Classical m6A-seq detects m6A in a sequence window from 3 to dozens of nucleotides^{[13, 3, 4, 5]}. Some improved methods (e.g. miCLIP-seq) achieve near single-base resolution by crosslinking m6A-antibody bound to the RNA to induced sequencing mutation (CIMS) or truncation (CITS) close to the modification site^{[12, 14]}. However, such CIMS or CITS patterns are complex, which can vary from one site to another and diffuse over a 3~4 bp window^{[12, 14]}.

Solution: Based on MazF digestion^[10], the RNA fragments with uncleaved m6ACA and the input RNA without MazF digestion are two-color labeled and then hybridized with Arraystar m6A Single Nucleotide Arrays, thus profiling the m6A level at single nucleotide resolution.

•  Lack of ​quantification ​​of m6A stoichiometry

m6A/MeRIP-seq does not ​quantify ​​the m6A stoichiometry, i.e. the fraction of m6A modification at that site. This missing information critically hampers functional prioritization of m6A sites and answering questions about the m6A writing/reading/erasing, regulation, and dynamics in response to stimuli^{[7, 15, 16]}.

Solution: The fraction or percentage of m6A modification can be quantified by the two-color hybridization intensities at each interrogated site, addressing the unfulfilled long-standing need in determining the dynamic m6A status.

•  Requirement of large RNA sample amount

m6A/MeRIP-seq requires massive RNA sample amounts (> 120 ug total RNA) ^{[3, 4, 12-14]}, ​which is prohibitive for samples of limited supply, such as precious clinical specimens, particular histological sites, or low yield sorted cells. While some protocols have been optimized for the purpose of substantially reducing the RNA amounts^{[17, 18]}, no methods exist for low RNA sample amounts while capable of m6A profiling at single-nucleotide resolution.

Solution: The microarrays use as low as 1 ug total RNA. The highly sensitive and specific MazF works very well even on extremely low RNA amounts at nanogram or picogram level. The rapid and simple techniques without immunoprecipitation dramatically reduce the RNA sample amount needed. m6A profiling can now be performed on rare samples, precious pathological specimens, particular histological sites, low yield sorted cells, or small animal models.

Table. Arraystar m6A Single Nucleotide Arrays vs MeRIP-seq

	m6A Single Nucleotide Arrays	MeRIP-Seq
Quantification	•  Modification stoichiometry as %Modified •  m6A RNA abundance •  Differential analysis of both %Modified and abundance	•  Lack of modification stoichiometry •  Differential analysis of abundance only
m6A-site resolution	•  Single-nucleotide	•  ~ 100 nt
Starting RNA amount	•  1 μg total RNA	•  120 μg total RNA
mRNA enrichment or rRNA removal	Not required	Required (Reaction scale-up needed)
RNA integrity demand	Tolerant, e.g. serum, plasma, exosomes, or FFPE samples	High

 

Related Service

m6A Single Nucleotide Array Service

 

References

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