RNAi

Question 1

important characteristics of RNAi in C. elegans (4)

Answer 1

• RNA must be double stranded
• dsRNA from introns and promoters don’t work, must be from the mRNA
• heritable
• may have cross-effects if multi-gene family has shared sequences

Question 2

what pathway did the mechanism of RNAi work out to be?

Answer 2

• the siRNA pathway

Question 3

what are the major differences between the miRNA and the siRNA (RNAi) pathway? (3)

Answer 3

• siRNA can be produced from longer dsRNA
• requires perfect homology with target
• directs cleavage of target mRNAs

Question 4

how is RNAi used in the cell? (2)

Answer 4

• defense against double-stranded RNA viruses

- regulation of protein expression

Question 5

how can RNAi be utilized in the lab

Answer 5

• used to target mRNA for degradation

Question 6

how is RNAi useful for reverse genetics

Answer 6

• can reduce gene expression for study without knocking out the gene entirely

Question 7

what are 2 means to generate double stranded RNA construct and what subsequent step must be made (3)

Answer 7

1. construct that can be transcribed in both directions
2. construct that is capable of forming a dsRNA hairpin with target sequences in the stem portion
   - must insert construct into a plasmid for generating dsRNA

Question 8

what methods can be used to introduce RNAi into C. elegans (3)

Answer 8

• injection
• soaking/feeding
• internal expression

Question 9

what are the steps for large-scale RNAi screens to identify genes and phenotypes in forward genetics (4)

Answer 9

1. clone diff genes/sequences into the RNAi vector
2. transform clones into E.coli and feed each to a C. elegan
3. identify C. elegans with phenotypes of interest
4. identify the gene that was knocked down

Question 10

what are the cautions of RNAi (2)

Answer 10

• RNAi might target more mRNA than intended, especially if it targets a conserved sequence; off-target effects
• mis-matched targets may lead to repression through miRNA pathway

Question 11

how can you prevent off-target effects of RNAi (2)

Answer 11

• use different sequences within the same target gene for the dsRNA; it is better if each dsRNA produces the same phenotype
• screen sequences to identify possible off-targets and avoid using dsRNA that have predicted off-targets