.Beginning with intact cells, explain in a step by step fashion how microarray e
ID: 218031 • Letter: #
Question
.Beginning with intact cells, explain in a step by step fashion how microarray expression profiles of non-coding RNAs in cancer cells as compared to normal cells can be determined. How would you validate the results obtained through microarray analysis? Is this the best approach to determine differentially expressed noncoding RNAs in these cells? What is the best approach to profile differentially expressed miRNAs and IncRNAs separately? How would you predict the functions of the differentially expressed miRNAs and IncRNAs in this experimental system?Explanation / Answer
-Tissue collection(both cancerous and normal) with intact cells-RNA extraction using TRIzol reagent and quantification using Nanodrop-mRNA is isolated and transcribed into fluorescent cDNA using Quick Amp Labeling kit-labeled cDNA purified,concentration and specific activity measured-1 microgram of each labeled cDNA is fragmented-hybridization buffer added-50 µl of the hybridization solution was dispensed into the gasket slide and assembled to the lncRNA expression microarray slide.
-validation-randomly selected lncRNA's were reverse transcribed and amplified using RT-qPCR,conditions were maintained as per the manufacturer's protocol.Some RNA's are upregulated and some are downregulated in cancer tissues.
-But this approach alone is not enough to determine differentially expressed noncoding RNAs in these cells.
-Gene Ontology(GO) and pathway analyses are used to determine the roles of differentially expressed mRNAs in biological pathways or GO terms. Differentially regulated mRNAs are uploaded into the Database for Annotation, Visualization and Integrated Discovery, which utilize GO terms to identify the molecular function represented in the gene profile. Pathway analysis is carried out based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
-To predict and understand the function of the targets of differentially expressed lncRNAs and miRNAs, GO terms and KEGG pathway annotation are applied in the present study to the target gene pool.
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