![]() Then, 15 μL of each reaction was processed within 24 hrs for PCR purification and sequencing was performed on ABI 3730 × l DNA Sequencers (Eton Bioscience, San Diego, CA). Production of amplicons of the predicted size was verified for each outer primer set by DNA gel electrophoresis using 10-20 μL of the completed reaction mixture separated on 2% agarose gels containing ethidium bromide and visualized under UV light. PCR cycling conditions were as follows: 95☌ for 2 minutes, followed by 35 cycles of 95☌ for 20 seconds, 62☌ for 10 seconds and 70☌ for 10 seconds, with a final extension step of 70☌ for 30 seconds. “Outer” primers (Additional file 2: Table S2) used for sequencing produce predicted amplicons of approximately 150-250 nucleotides in length, and flank each editing site with approximately 50-100 bp on either side of the editing site to facilitate successful sequencing analysis. Traditional Sanger sequencing is not sufficiently sensitive to detect editing events in rare stem cell regulatory transcripts, and transcriptome-wide profiling of RNA editing can be costly, technically challenging, and analysis requires expertise in specialized bioinformatics methods.įor PCR and targeted Sanger sequencing analysis, 1-2 μL of first-strand cDNA templates were prepared for PCR in 25-50 μL reaction volumes using the high-fidelity KOD Hot Start DNA Polymerase kit according to the manufacturer’s instructions (EMD Millipore, Temecula, CA). However, the functional role of RNA editing of individual transcripts, and its role in cancer progression and drug resistance, has not been widely addressed due to a lack of tools to quantify functionally relevant RNA editing events in a sensitive, cost-effective manner. Since RNA editing may be selectively inhibited, it is of great clinical relevance to develop diagnostic and prognostic tools capable of accurately detecting fingerprints of aberrant RNA editing activity signifying cancer progression and therapeutic resistance. Previously, we found that human leukemia stem cells (LSC) from patients with blast crisis (BC) chronic myeloid leukemia (CML) harbored increased expression of ADAR1 compared with normal and chronic phase (CP) progenitors. With the availability of such massive new datasets, it is now critical to apply this knowledge to mine new and existing RNA-sequencing (RNA-seq) datasets of human tissues, to identify disease-relevant RNA editing loci. Additionally, aberrant RNA editing may drive stem cell regulatory transcript recoding and microRNA deregulation leading to therapeutic resistance.Īdvances in next-generation sequencing technologies and bioinformatics tools have led to the identification of hundreds of thousands of RNA editing sites throughout the human transcriptome, the majority of which are localized to hyper-edited regions. While RNA editing targets are relatively conserved in normal tissues, CSC-associated editing changes in response to malignant microenvironments could dramatically alter gene product stability and function. At the transcript level, RNA editing can affect mRNA stability, localization, nuclear retention, and alternative splicing. In mouse hematopoietic development, ADAR1 plays a key role in hematopoietic stem cell (HSC) survival and cell fate determination, and ADAR1 is the primary RNA editase expressed in human hematopoietic stem and progenitor cells. The ADAR family consists of three members, ADAR1 ( ADAR), ADAR2 ( ADARB1), and ADAR3 ( ADARB2). Most RNA editing is carried out by ADAR-mediated C6 deamination of adenosines (A) to inosines (I). ![]() Of particular relevance to the pathogenesis of human disease, over 90% of RNA editing occurs in primate-specific Alu sequences that form dsRNA secondary structures, often within non-coding regions such as introns and 3′UTRs. Detection of a CSC-specific RNA editing fingerprint of leukemic progression.Detection of lentiviral ADAR1-induced RNA editing in primary human cells.RNA editing site-specific qRT-PCR (RESSq-PCR) assay design and validation.Development of an in vitro model of ADAR1-dependent RNA editing.Selection and validation of aberrant RNA editing events associated with leukemia progression.Nucleic acid isolation, reverse transcription and quantitative RT-PCR.Generation of a stable ADAR1 RNA editing detection model system.Transduction of human cell lines and primary cells with lentiviral-ADAR1.Lentiviral vector preparation and ADAR1 site-directed mutagenesis.High-fidelity PCR and Sanger sequencing analysis.
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