Methods for in‐depth genome‐wide characterization of transcriptomes and quantification of transcript levels using various
microarray and next‐generation sequencing technologies have emerged as valuable tools for understanding cellular physiology
and human disease biology and have begun to be utilized in various clinical diagnostic applications. Current methods, however,
typically require RNA to be converted to complementary DNA prior to measurements. This step has been shown to introduce many
biases and artifacts. In order to best characterize the ‘true’ transcriptome, the single‐molecule direct RNA sequencing (DRS)
technology was developed. This review focuses on the underlying principles behind the DRS, sample preparation steps, and the
current and novel avenues of research and applications DRS offers. WIREs RNA 2011 2 565–570 DOI: 10.1002/wrna.84
Illustration of direct RNA sequencing sequencing preparation procedure. Poly‐adenylated and 3′ blocked RNA is captured on surfaces containing covalently bound poly(dT) oligonucleotide (3′ end of the poly(dT) oligonucleotide faces ‘up’). A ‘fill and lock’ step is performed, where the ‘fill’ step is performed with natural thymidine and polymerase, and a ‘lock’ step is performed with fluorescently labeled A‐, C‐, and G‐Virtual‐Terminator nucleotides and polymerase. These steps correct any misalignments that may be present in polyA and polyT duplexes and ensure that the sequencing starts in the RNA template rather than the poly‐adenylated tail. Imaging is performed to locate the positions of the templates.
Diagram of single‐molecule sequencing instrument optics. A 635‐nm laser is used to illuminate the surface through the objective lens using total internal reflection. This generates an evanescent wave that results in a restricted excitation field, important for the reduction of background fluorescence. Fluorescent single molecules within the excitation field on the flow cell surface emit light, which is captured by the objective lens and detected by the charge‐coupled device camera.
and her research team focus on the dissection of the molecular mechanisms and pathways involved in Lin28-mediated regulation. First, they will analyze Lin28 expression in mouse and human ES cells to determine whether its expression is regulated during the cell cy-cle. Then, they will characterize the interactions between Lin28 and its associated mRNAs to gain molecular insights into their assembly, function and regulation in the cellular milieu. Finally, they will strive to identify Lin28-interacting protein partners and new target mRNAs to establish a comprehensive and global understanding of Lin28 function.