RNA methylations play a significant regulatory role in diverse biological processes. Nowadays, the importance of RNA modifications and RNA methylation in particular is evident. To date, 171 RNA modifications are known according to the MODOMICS database, of which 72 include methyl groups . The internal modifications are present in different RNA classes, such as tRNA, rRNA, mRNA, snRNA, lncRNA as well as in viral RNA genomes. The biological functions of RNA methylation greatly vary depending on the modified nucleoside and the RNA type. Several modifications, including N6-methyladenosine (m6A), 5-methylcytidine (5mC), inosine (I), pseudouridine (Y), N1-methyladenosine (m1A) and 5-hydroxylmethylcytidine (5hmC), are found internally in eukaryotic mRNA and can influence the metabolism and function of mRNA. Recent years have witnessed major advances in the development of novel transcriptome wide sequencing technologies for distinct methylation (epitranscriptomic) marks. These new tools have helped researchers to identify the location of RNA modifications and to reveal these modifications’ distinct distribution patterns throughout the transcriptome.
- Type of chemical modifications in mRNA:
Quick Biology is offering two types of RNA methylation sequencing service for 5mc and m6A:
(1) 5mC RNA Bisulfite-Seq
- A complete and optimized workflow designed for easily carrying out RNA bisulfite conversion of 5-methylcytosine, followed by a "post-bisulfite" library preparation process for Illumina platform-based bisulfite sequencing, all in one go. Our comprehensive service offer: (1) High sensitivity and efficiency (provides a robust and reliable means to build 5mC RNA-seq library), (2) Complete conversion (converts unmethylated cytosine into uracil at a level greater than 99.9%), (3) Minimal bias (RNA libraries with minimal sequence bias and low error rates) and (4) Broad sample suitability (from RNA isolated from various tissue/cell samples such as fresh and frozen tissue, cultured cells from a flask or microplate).
5mC RNA Bisulfite-Seq sample requirements:
· The amount of RNA for each bisulfite reaction can be 5 ng to 500 ng. For an optimal reaction, the input RNA amount should be 100 ng to 200 ng.
· Starting materials can be total RNA isolated from various tissue/cell samples such as fresh and frozen tissues, cultured cells from a flask or microplate, microdissection samples, and body fluid samples, etc.
(2) m6A RNA-seq (MeRIP-seq, Methylated RNA Immunoprecipitation)
- Methylated (m6A) RNA immunoprecipitation (MeRIP) is a method to monitor the status of m6A and map the location of m6A RNA modifications transcriptome-wide. Quick Biology uses the MeRIP method to enable identification and transcriptome-wide profiling of m6A RNA modifications. In the MeRIP assay, RNA is chemically fragmented into 150 nucleotides or smaller fragments followed by magnetic immunoprecipitation with a monoclonal antibody toward m6A. After immunoprecipitation, isolated RNA fragments can be subjected with RNA sequencing (MeRIP-seq). Our streamlined protocol is easy to process and can produce results with high Signal to Noise ratios.
m6A RNA-seq sample requirements:
· Started from total RNA, mRNA, or rRNA depleted RNA.
· > 0.5 ug of mRNA or > 30 ug of total RNA per immunoprecipitation
· Use of intact RNA is critical. We recommend to use column purification and check the RNA integrity by Bioanalyzer. Degraded RNA affects to the quality of the assay.
· DNase treatment is highly recommended. This is more important working with RNA from organism known to contain m6A in their DNA. The anti-m6A antibody recognize m6A in single strand DNA.
- In-Depth Data Analysis is also available, please contact us for details.
About 4 weeks, depends on the quality of submitted samples, one additional week for data analysis.