A spliced 2

A spliced 2.2-kb RNA transcript was first identified in transfected hepatoma cells [77] and contains a single 1, 223-nt long intron starting from the end of core antigen ORF to the middle of S antigen ORF. primer pair and a PCR machine are needed in a lab setting and identification of a viral pathogen takes within few hours. They are sensitive and specific and require only a small amount of patients materials to detect a specific nucleotide sequence region. In general, these techniques can be used to detect almost all types of viral pathogens and even to identify multiple viral pathogens or their variants at the same time. In this chapter we focus on detection of viral RNA splicing as a new tool for diagnostic virology. and below indicate positions of antisense probes commonly designed for northern blot to detect products by each probe. N/D not detectable. (b) Detection of spliced RNA products by RPA. Diagram shows an antisense riboprobe spanning over an intron region (a or polymerase extends the primer to synthesize the nascent strand, the 5C3 exonuclease activity of the polymerase degrades the represent a double-stranded DNA template generated by RT-PCR while a is correspondent to single-strand DNA. mark primer (and represents quencher. Scorpions probes contain amplification stop sequences named blocker (B in polymerase inactivating contaminants in clinical samples, and amplification bias. Other considerations are false positivity and PCR cross-contamination. Splicing Microarrays DNA microarrays (also known as DNA chips) are composed of large number of probes, often in several thousands, spotted on very small area in 2D format on solid surface (glass or plastic). Probes represent DNA oligos of various length and chemistry. Each probe has specific DNA sequence allowing detection of corresponding DNA with complementary sequence. Currently, there are two major technologies of DNA arrays manufacturing of microarray: (a) direct Jatropholone B synthesis of probes on the array or (b) printing arrays from library of presynthesized probes. Each DNA microarray allows rapid profiling of large Jatropholone B number of DNA molecules at the same time. Today DNA microarrays are widely used to study gene expression profiling and RNA posttranscriptional modifications including RNA splicing [21]. The analysis of RNA transcripts by DNA microarrays requires a conversion of RNA samples to DNA by reverse transcription, following amplification and labeling with fluorescent dye. After labeling the samples are hybridized with the probes on the array. Unbound samples are washed away and the fluorescent signal is captured and analyzed by microarray readers (Fig. 38.6a). The intensity of fluorescent signal corresponding to the number of bound molecules allows determination of the level of RNA in the original sample by mathematical algorithm. Open in a separate window Fig. 38.6 Splicing microarrays and in situ hybridization. (a) A work flow for microarray assay. First, sample RNAs are converted into cDNAs simultaneously labeled with specific fluorophore (F). The labeled cDNAs are hybridized with oligonucleotide probes attached to a solid surface. The unbound cDNAs are washed away and the remaining fluorescence signals resulted from specific hybridization are collected by Jatropholone B an array-scanning device and analyzed. (b) Microarrays in RNA splicing detection. (i) Tiling arrays represent a large set of overlapping probes (provides a comprehensive picture of whole genome transcriptome and has been successfully used for analysis on gene expression and posttranscriptional processing including RNA splicing. However, NGS is costing and requires sophisticated data analysis, which makes it less suitable for clinical diagnostics. However, does not require any prior knowledge of detecting sequence composition and therefore allows to detect unknown or unpredicted RNA sequences. This may be especially beneficial in discovery of new pathogens including viruses [34]. In addition, instantly analyzes a transcriptome including spliced transcripts of any type of cells or tissues. RNA Splicing in Mouse monoclonal to LPP Clinical Virology RNA splicing does not occur in prokaryotes and is a hallmark of the eukaryotic gene expression. In eukaryotes the number of genes which undergo splicing varies highly from organism to organism, with only about 5 % Jatropholone B of all genes being spliced in yeasts to 95 % in human [35, 36]. Viruses as intracellular parasites replicate inside of host cells and hitchhike many cellular processes for their multiplication including RNA splicing. By using constitutive and/or alternative RNA splicing, most of DNA.