Week 9 - Gene Expression part 1 Flashcards
Detection methods of mRNA?
Northern Blotting
ISH (In situ hybridisation)
RT-PCR/qPCR
Transcript profiling
Reporter plasmids/genes
What is northern blotting?
It is the separation of RNA by gel electrophoresis, It detects when bands anneal to the probe, annealing of a band to a probe indicates gene expression.
What 2 probes are used in northern blotting?
Sense - hybridises to DNA and cDNA and not mRNA
Anti-sense - hybridises to mRNA
Explain some other factors of Northern blotting?
Formaldehyde is used to remove RNAs secondary structure, The pH is maintained around neutral or slightly acidic as alkaline conditions can destroy RNA, RNA is fixed to membrane via UV treatment.
FISH (fluorescent in site hybridisation)
Detection and localisation of RNA in samples, samples need to fixed and DNA removed, labelled nucleotide probes are used to detect target RNA molecules.
q-PCR
used to monitor and quantify specific mRNA targets
q-PCR - non target sequence specific methods
Requires absolute specificity primers for their target sequence:
intercalating dyes - fluoreseces strongly when bound to dsDNA, fluorescence is measured during polymerisation
qPCR - sequence specific methods
Taqman, Molecular Beacons. Primers and probes are fluorescently labelled to allow detection, can be multiplexed - test for the presence of different templates
General principle of qPCR
requires cDNA, cDNA is amplified by PCR in presence of fluorescent stain, products are measured by monitoring the increase in fluorescence
q-PCR molecular Beacons
short oligonucleotides that gives off a fluorescent signal when it hybridizes to PCR product, caused by the dye and compound moving away from each other after annealing to PCR product
explain reporter genes
fusion of a gene promoter to a promoter less reporter gene allows effects on gene expression to be followed, as the reporter gene will mimic the expression of the promoter
