L21 - functional genomics - transcriptomics Flashcards
(12 cards)
what are the 3 ‘omes’
genome , transcriptome and proteome
what are functional genomics about
defining one or more of the ‘omes’ for a cell at a given point in time
= and so why it expresses a particular phenotype
what process do functional genomics rly on
hybridisation
= annealing of single-stranded labelled nucleic acids to targets
form of quantifying how much of target is there
= strength of signal is proportional to how much of target DNA/RNA is there
what does northern blotting meausure
RNA/Transcript levels
- isolate RNA and run on gel
- transfer RNA to nylon membrane via membrane blotting
- add labelled DNA probe that hybridises too target sequence
- wash surface so probe is washed off if RNA not present
- quantify signal/fluorescence and run on gel against control to ensure its the correct RNA
what do Western, Northern and eastern blotting look for
Western = proteins
Northern = RNA
Southern = DNA
Northern blotting uses mRNA in solid phase (nylon membrane) and DNA probe in liquid phase - this is very slow, how do we imporve this?
array approach
DNA probe is attached to solid phase and mRNA is the added liquid
= microarray technology
what are the problems with having mRNA as the liquid phase in a microarray approach AND how do we solve this
mRNA must be labelled
= mRNA is unstable and is difficult to label
solution:
convert mRNA into cDNA incorporating the label WHILE doing this with labelled primers
describe what oligonucleotide microarray technology is
has a glass slide wit thousands of tiny spots
= small known DNA sequnces/oligonucleotides are ‘sprayed on’ by a ink printer
flourescently labelled cDNAs made from mRNA with reverse transcriptase are added to the array
= hybridise and show colour in spot specific to that gene
compare and contrast northern blotting and oligonucleotide microarray technology
Both measure levels of RNA in cell/transcriptomics with fluorescently labelled probes
Northern blot:
detect and quantify specific RNA/gene expression
RNA in solid phase and labelled DNA in liquid phase added
= fluorescent band
Microarray:
measure expression of thousands of genes at once
uses labelled cDNA as liquid phase and short known DNA sequences as solid
= coloured spots
can you compare 2 samples at once with microarray experiment
yes
one sample is flourescently labelled with green probe and one with red when creating cDNA
cDNAs are both added to microarray
= the colour shown in each spot shows which samples cells contained more of that mRNA/had higher expression
name 2 problems with transcriptome profiling (microarray)
- semi-quantitative = you get a rough idea but no exact ‘quantitative’ number
- intensity at a specific spot is only ‘relative’ compared to the other sample
= this des not mean expression is especially high or low in either
describe RNA-seq
RNA sequencing is a powerful tool that uses next-gen sequencing to see the gene expression/mRNA level in a cell at a given time
- isolate mRNA and convert into cDNA with adaptor sequences at end
- stick to surface and add ddNTPs
= illumina sequencing
compare the reads/sequences identfied to the genome
= shows which genes were tuned on
= the reads shwon correlate to level of mRNA/gene expression in cell
