CRNA synthesis

In order to detect which sequences on the array (the probes ) have been hybridized by the sequences in the sample (the targets ), the later sequences have to be previously labeled. This is done by incorporating fluorescently labeled nucleotides in the cDNA synthesis step (in two-color arrays) or the incorporation of a biotin-labeled nucleotide in the cRNA synthesis step, as done by Affymetrix (47). 

Protocol dependent cRNA synthesis Bioanalyser (optional)... 

High levels of protein expression are generally achieved with cRNA injection. This technique requires the in vitro synthesis of the appropriate cRNA from the template cDNA (see later). Although this approach can be time-consuming and costly, it remains the most common technique used to ensure the robust expression of receptors and ion channels in the oocyte membrane. In our laboratory, we routinely use the cRNA injection technique to promote high expression levels of LGIC receptors (GABAa receptor subtypes, nACh and 5-HT3 receptors). 

A prerequisite for cRNA transcript synthesis is the presence of the cDNA of interest within a suitable expression vector containing a bacteriophage polymerase promoter (e.g. T3, T7 or SP6). The vector must be linearized downstream of the cDNA insert prior to its use as the template in the in vitro reaction. 

Budding and release of progeny virus. B. Replicative cycle of an influenza virus, an example of an RNA virus. 1. Attachment. 2. Endocytosis. 3. Influx of H+ through M2 protein. 4. Fusion of the viral envelope with the endosomes membrane, dissociation of the RNP complex, and entry of viral RNA into the nucleus. 5. Synthesis of viral mRNA by viral RNA polymerase. 6. Translation of viral mRNA by host cell s ribosomes. 7. Replication of viral RNA, using viral RNA polymerase, via cRNA replicative form. 8. Assembly of virus particles, and 9. Budding and release of progeny virus. 

In the case of the WT models, the labeling during cRNA in vitro transcription is not executed. Instead, there is a second cycle of cDNA synthesis and random priming amplification from the cRNA strand, with a subsequent cRNA hydrolysis. In this case, the cDNA is the labeled molecule that will be hybridized against the surface attached probes. While using the same basic chemistry as its predecessors, it has been optimized to allow for lower sample input and faster processing times, while retaining comparable result quality and reproducibility. 

The result is high-quality arrays that can be used with a fluorescence detection system in a manner similar to the glass slide microarrays, although only single color detection is used. cDNA is synthesized from a test RNA sample by reverse transcription followed by second strand synthesis. This is then used as a template for an in vitro transcription reaction to produce biotin-labeled cRNA. As well as introducing the label for detection, this step permits amplification of the RNA and thus enables less starting material to be used. The cRNA is hybridized to the GeneChip and the biotin detected with an avidin-... 

If there is to be a long duration between synthesis of the cRNA and hybridization to the microarray, the biotin-labeled cRNA should be stored prior to fragmentation. 

Rakusanova et al. (1972) examined the pulse-labeled RNA that annealed with cell DNA (cRNA). Both production and turnover of nuclear cRNA were inhibited in cells infected with PRV. Only 10% of the cRNA detected in the cytoplasm of infected cells was found associated with a polysome fraction as compared to 40% in normal cells. The remaining 90% of cytoplasmic cRNA that was not associated with polysomes nevertheless contained poly(A). The implication is that both the synthesis and processing of cellular mRNA were interrupted in infected cells and that those molecules that entered the cytoplasm, although carrying poly(A), were not able to as-... 

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