Project description:Methods that enable absolute quantification of N6-methyladenosine (m6A) RNA modification have emerged as powerful tools in the field of epitranscriptomics. We previously reported GLORI, a chemical-assisted approach firstly achieved quantitatively transcriptome-wide m6A measurement at single-base resolution. Despite its advantages, GLORI suffers from lengthy reaction time and severe RNA degradation. Here, we present two updated GLORI approaches: GLORI 2.0 is an ultra-fast and mild version that preserves RNA integrity and enhances sensitivity for both transcriptome-wide and locus-specific m6A detection; GLORI 3.0 further utilizes a novel reverse transcription-silent carrier RNA to achieve high-quality m6A quantification from ~ 1,000 cells. Using limited RNA input extracted from single mouse dorsal hippocampus, we measure m6A methylome in the synaptic and cytoplasmic fractions and reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m6A in biology.
Project description:We developed a quantitative method called GLORI to investigate m6A methylation in the mammalian transcriptome at single-base resolution.
Project description:To investigate the m6A methylation in the mammalian transcriptome, we developed a quantitative method, names GLORI, that could detect m6A stoichiometry at single-base resolution. We then performed GLORI on cells with different treatment, such as stress, knockdown and inhibitor. We next analysis m6A methyloms of different celllines and cells under different treatment to investigate the fuctional role of m6A.
Project description:GLORI-Seq is an antibody-independent sequencing, which can detect m6A sites at single-base. The core of GLORI-Seq is to find a catalytic system of glyoxal and nitrite through chemical reaction combination screening, which efficiently deaminate unmethylated adenosine A to form inosine (A-to-I, > 98%). Inosine is read to guanosine (G) in the sequencing process, forming A-to-G conversion. GLORI-Seq enables single base detection of single base m6A by detecting the proportion of “A” in the sequenced read sequence.
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells.