CRM RNA Flashcards
cricks central dogma on genetic flow outline
DNA to RNA to protein to function
what are some changes that can occur during flow of genetic information
DNA- SNP and genetic variants reverse transcription between DNA RNA epigenetics RNA alternate splicing miRNA protein post translational modification
transcriptome(all expressed genes- proteome too diverse and complex)
not fixed, responds to stimuli in health and disease
working with RNA
uracil not thymine
single unstable strand
secondary and tertiary structures
no introns (spliced out in processing)
some examples of applications of RNA
northern blots in situ hybridisation qRT-PCR TaqMan cDNA libraries(cDNA) gene expression microarrays(cRNA)
how to use cDNA libraries
incorporates bacterial plasmids giving rise to colonies to be sequenced
gives idea of which genes were expressed in source material at time of isolation
cDNA libraries application
identify novel genes and splice variants, can also generate template for full length cDNA analysis
cDNA info
complimentary DNA
no intronic sequence or non coding regulatory sequences
cDNA smaller than full genetic sequence so less resource intensive than screening whole DNA
using northern blots
mRNA seperated using GEP
RNA blotted onto membrane
incubated with radioactive labeled probes complimentary to genes of interest
prayed to see bands of radiation to give idea of level of gene expression
northern blot applications
see expression levels
bio distribution of gene
presence of splice variants
sensitive and specific
downsides of northern blots
being replaced by newer techniques with higher sensitivity and without the use of toxic materials
in situ hybridisation
Difficult technique
similar to norther blot but use this section of tissue/cell/organism
in situ hybridisation how it works
labelled probe detects mRNA and allows provide localisation and gives indication of abundance
gene expression microarrays why its used
get a quantitative snapshot of expression level of the entire genome at a specific time point in a given tissue or cell type
gene expression microarrays problems
expensive and generates lots of data
microarray application
used to identify targets then use TaqMan to find out about protein and function
used to see what genes are related to cancer and type of cancer eg chemo sensitive cancers
quantitative real time PCR (qRT-PCR)
having identified a gene/genes PCR quantifies amount of ds-nucleic acid
amplifies genes of interest incorporates fluorescent markers into DNA
qRT-PCR analysis
using serial dilutions absolute quantification
use a control group relative quantification
miRNA what is it an how it works
short non coding negative regulators of gene expression by inhibiting mRNA translation or promoting mRNA degradation
important in diseases such as cancer
miRNA biogenesis
primiRNA to premiRNA by RNase Drosha
premiRNA exported out nucleus into cytoplasm by exportin 5
premiRNA cleaved by dicer into mirna duplex then assembled into mature miRNA
miRNA and examples of disease
induction of heart failure
cardiac hypertrophy and wall thinning
miRNA therapeutic potential
can regulate pathways and have sustained effects, but hard to deliver, may have off target effects and could be pathogenetic
