Lecture 10: Transcriptomics Flashcards
Describe the process of protein production from genes
Genes are encoded by DNA and are transcribed to produce messenger RNAs (mRNAs) which are translated to produce proteins.
What is Southern blotting used for?
Used for detecting DNA
Describe the process of Southern blotting
Native DNA fragments are separated on the basis of size by gel electrophoresis and then denatured and transferred to a membrane. A specific labelled DNA fragment is used as a probe to detect specific fragments in the DNA sample.
What is Northern blotting used for?
Used for detecting RNA
Describe th process of Northern blotting
A denatured RNA sample is separated on the basis of size by gel electrophoresis and transferred to a membrane. Remains denatured during electrophoresis so mobility is proportional to size. A specific labelled DNA fragment is used as a probe to detect specific transcripts in the RNA sample.
Describe the process used for the detection of gene expression by microarrays
DNA is transcribed to form mRNA and then labelled using Cy3 or Cy5-modified dNTPs. Reverse transcriptase catalyzes the generation of Cy-labelled cDNA which is then added to arrays. Random hexamers are used to prime the cDNA synthesis.
A reference sample is co-hybridised with the test sample, each labelled with a different dye (fluorochrome).
Microarray is then laser scanned to aid in detection of similarities and differences between the test sample and the reference sample. Machine generates coloured spots on a glass film which enables the calculation and generation of a gene expression matrix (GEM) to determine expression levels of particular genes.
What is the use of microarrays?
DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in its genes. Aids in the detection of deletions/amplifications and other allelic variations in genomes; comparative genomic hybridisation (CGH).
What is a gene expression matrix (GEM)?
Grid where each row represents a gene and each column represents a sample. Each entry in the matrix grid represents the expression level of a particular gene in a given sample (cell).
What are gene expression profiles (GEP)?
Measurement of the transcriptional activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can distinguish between cells that are actively dividing, or show how the cells react to a particular treatment.
Wht are the purpose of DNA microarrays?
Measure the relative activity of previously identified target genes
How do sequence-based transcriptomic techniques differ from simple microarrays?
Sequence based techniques, like RNA-Seq, provide information on the sequences of genes in addition to their expression level, whereas microarrays only identify expression levels.
How does RNA-seq work?
Isolates total RNA, removes contaminating DNA, and removes ribosomal RNA (by selective capture with antisense oligos). Uses fragment RNA to generate cDNA by use of reverse transcriptase enzyme. Adaptors are then added to allow for attachment to a flow cell, and then normal next-generation sequencing, ie. Illumina, is conducted to identify the DNA sequence for the gene/ fragment.
What are the advantages of the RNA-seq technique?
- Enables identification of novel RNA transcripts that may not be printed on the microarray – since RNA-seq reads cDNA from all RNA molecules in the sample.
- Enables mapping of transcripts at single nucleotide resolution allowing identification of 5’ and 3’ ends.
- Provides more precise information on different RNA splice variants
How can genomics be used in healthcare?
Genomics can help guide targeted treatment for many diseases and types of cancers. Genomics can help enable the deduction of which cellular pathways are perturbed, for particular diseases, or which genes have altered expressions, for particular cancers, to help design targeted therapy, ie. For example with monoclonal antibodies.
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