- learn about luciferase assay
Paper describing how to design a miRNA:
- "In general, we target the coding region; however, targeting the 5′- and 3′-UTR sequences is possible"
- "It is important to note that siRNA design rules serve more as guidelines, and that sequences adhering to them may not silence and vice versa. To date, no algorithm guarantees silencing efficacy, and most recommend the user pick three to four candidates for screening."
- "To achieve faithful loading of the antisense strand, the duplex must be designed such that there is strong G–C base pairing present at the 5′-end of the sense (passenger) strand and weak A/G-U base paring at the opposing terminus"
Questions/concerns about miRNA:
- "Overall hairpin length and loop size influence the efficiency of Dicer processing"–how should we design the loop?
- The specificity of base pair matching in the stem influences processing efficiency
- Which of the two strands from the stem is incorporated into the RNA-induced silencing complex?
- miRNA's are readily degraded by RNase (present ubiquitously in cells). So we should be sure to inhibit RNase when working with miRNA's; this inhibitor is available for purchase or might already be in the lab.
Pay attention to cross talk. look for foreign miRNA
BLAST NCIB