RNA Flashcards
What are the key features of RNA?
Ribonucleic acid Ribose-phosphate backbone 4 nucleic acid bases single stranded (not as stable) many subtypes can adopt secondary and tertiary structures no introns 5 prime cap and 3 prime poly-A tail
What are some considerations of working with RNA?
Good preparation is vital Readily degraded Sterile equipment - RNAse ZAP work "on ice" small volumes, good pipettes, competence
Why is cDNA synthesised?
double stranded - can be used for PCR
Preserves RNA sequence
more stable
can select target RNA
What are cDNA libraries?
incorporated into bacterial plasmids, resulted colonies picked and sequenced
gives snap shot of gene expression in source of isolation
What are some applications of cDNA libraries?
ID novel genes
ID splice variants
Generate template for full cDNA analysis
What are the pros and cons of cDNA libraries?
No intronic sequences
smaller - easier
less resource intensive than sequencing genomic DNA library
What are the applications of Northern Blots?
determination of bio-distribution of expressed gene
Determination of size of transcript
presence of splice variants / processing mRNA
Estimation of expression levels
What are the pros and cons of Northern Blots?
sensitive specific membranes stable over months use toxic materials now newer techniques with greater sensitivity
Describe in-situ hybridisation
conceptually similar to northern blots but uses tissue or cells
labelled probe detects target mRNA and allows precise localisation of target, as well as giving indication of abundance
Analogous to histology
similar pros and cons to northern blots
notoriously difficult to do
What is miRNA?
short, non-coding RNA molecules that act as negative regulators of gene expression by inhibiting mRNA translation or by promoting mRNA degradation
What is the therapeutic potential of miRNA?
can target many mRNA molecules
Can regulated multiple functionally related mRNAs - to regulated complex pathways
What are the advantages of targeting miRNA?
can regulate multiple components of same pathway or cellular process
long-lived sustained effects
target miRNA is defined
effective in vivo regulation of mRNA
What are the challenges of targeting miRNA?
delivery of miRNA modulator
off-target effects
beneficial and pathogenic effects
toxicity of miRNA modulator
