Transcriptomics

Transcriptomics

1. Northern Blot

   • Analytical technique
   • Studies gene expression by detection of RNA in a sample
   • Useful to observe cellular control over structure and function by determining gene expression levels in abnormal (e.g. differentiation, morphogenesis) and diseased conditions
   • Electrophoresis
     • Separates RNA samples by size
       • Detection with a hybridisation probe
         • Probe is a section of nucleotides complementary to part of (or the entirety of) the target sequence
   • Time consuming and challenging

2. Microarrays

   • Most common method of transcriptomics
   • Carries out many genetic tests at the same time, for thousands of genes in parallel
   • mRNA molecules for each gene of interest are collected for both the 'normal' and 'abnormal' cell
     • Hybridisation probes for the genes of interest are placed onto a microarray slide
       • cDNA molecules are then generated from the mRNA
         • Each sample is labelled with fluorescently labelled nucleotides
           • Normal and abnormal cDNA molecules are labelled with different colours
         • 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
         • 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
   • Example
     • Study of tumour cells
     • Tumour cells are labelled with a red dye, and normal cells are labelled with a green dye
     • When co-hybridising samples together, they compete to bind to the complementary probe
     • 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
     • Duplication of the nucleotide sequences in the genome is a real problem
       • 37% of genes are highly conserved, with a further 10% being partially conserved, meaning that a labelled target sequence could hybridise to different probes
       • Better solutions are gene-specific tags, or using a GeneChip to quickly determine which genes are present in a sample

3. Mass Transcript Profiling