Transcript RNAi - Mabra

RNAi
miRNA and siRNA
DNAProtein
• DNA  RNA
• RNAProtein
http://www.nobelprize.org/educational/medic
ine/dna/index.html
mRNA
• The instructions for making protein
• If you can destroy the mRNA the protein won’t
be made
• How could you destroy only the harmful
mRNA without hurting all the other RNA in
the cell (the cell is making many useful
proteins)
HINT1: How does this relate to
viruses?
• Some viruses have double stranded RNA
• To survive, human cells had to destroy all
double stranded RNA in the cell (the normal
cell doesn’t have any)
• Use something that is already inside the cell!
HINT 2:
• Remember! Viruses make copies of their
DNA/RNA inside the host cell- destroying one
copy is not enough
HINT 3
• Think about complementary pairs!
The answer!
• siRNAs- silencing RNAs
• 1) Insert a double stranded piece of RNA with
one piece complementary to the RNA you
want to destroy
• 2)The cell will destroy this siRNA, but first it
will take one side (single stranded RNA) and
march around the cell destroying any RNA
that matches that RNA
RNAi
• http://www.nature.com/nrg/multimedia/rnai/
animation/index.html
How can this be used for treatment of
cancer?
Practical side of RNAi
• http://www.invitrogen.com/site/us/en/home/
References/Ambion-Tech-Support/rnaisirna/tech-notes/rnai-how-to--for-newusers.html
Scientific Techniques
• You have to insert a dsRNA that is less than 30
base pairs long, because 30+ base pairs makes
the cell mount a much stronger antiviral
response
• The RNA should match exactly the gene you
want to silence
From Invitrogen
Reagents Required for RNAi Experiments
The reagents required for inducing and
analyzing the RNAi effect are quite simple:
• A specific dsRNA that targets a particular gene
transcript to induce the RNAi pathway
• An efficient dsRNA delivery system
• Assays for the RNAi effect
• Proper controls
specific dsRNA that targets a
particular gene
• Must know the sequence
• Can synthesize RNA chemically
efficient dsRNA delivery system
• Immortalized cell lines: “transfection with a
lipid- or amine-based reagent is the preferred
option”
• Other cells: “electroporation using a
specialized, gentle-on-cells buffer and
optimized pulsing conditions generally results
in very efficient siRNA delivery without
compromising cell viability”
Control Cells
• Cells that the “silenced” cells can be
compared to
• What should you do?
Assays for the RNAi effect
• Need: something that monitors the mRNA
levels in the cell
• Want: Something that measures gene activity
– Concentration of enzyme
– A certain metabolic pathway
This seems REALLY difficult
“Ambion, in partnership with Cenix BioScience,
provides expert designed, guaranteed-tosilence siRNAs to >34,000 human, mouse, and
rat targets (>98% of all human, mouse, and rat
genes in the RefSeq database).”