Transcriptomics

Northern Blot
1. Analytical technique
2. Studies gene expression by detection of RNA in a sample
3. Useful to observe cellular control over structure and function by determining gene expression levels in abnormal (e.g. differentiation, morphogenesis) and diseased conditions
4. Electrophoresis
  1. Separates RNA samples by size
    1. Detection with a hybridisation probe
      1. Probe is a section of nucleotides complementary to part of (or the entirety of) the target sequence
5. Time consuming and challenging
Microarrays
1. Most common method of transcriptomics
2. Carries out many genetic tests at the same time, for thousands of genes in parallel
3. mRNA molecules for each gene of interest are collected for both the 'normal' and 'abnormal' cell
  1. Hybridisation probes for the genes of interest are placed onto a microarray slide
    1. cDNA molecules are then generated from the mRNA
      1. Each sample is labelled with fluorescently labelled nucleotides
        Normal and abnormal cDNA molecules are labelled with different colours
      2. Labelled cDNA molecules hybridise to their complementary probes on the slide, leaving their fluorescent tag
        A scanner can be used to measure fluorescence intensity for each spot on the slide
      3. If a particular gene is very active, it produces more mRNA, and therefore more cDNA
        This means more molecules hybridise to the slide, generating a very bright fluorescent spot
        Less active genes produce a dimmer fluorescent spot
        Inactive genes produce no mRNA, so there is fluorescence
4. Example
  1. Study of tumour cells
  2. Tumour cells are labelled with a red dye, and normal cells are labelled with a green dye
  3. When co-hybridising samples together, they compete to bind to the complementary probe
  4. As a result, a red spot means that the gene is more expressed in cancer cells (up-regulated in cancer), and a green spot means that it is more expressed in normal cells (down-regulated in cancer) and a yellow spot means that the gene is equally expressed in normal and cancer cells
  5. Duplication of the nucleotide sequences in the genome is a real problem
    1. 37% of genes are highly conserved, with a further 10% being partially conserved, meaning that a labelled target sequence could hybridise to different probes
    2. Better solutions are gene-specific tags, or using a GeneChip to quickly determine which genes are present in a sample
Mass Transcript Profiling

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Transcriptomics

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	Erstellt von Lydia Buckmaster vor fast 11 Jahre