20.109(F07):Module 2: Difference between revisions
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In this experiment, we will consider | In this experiment, we will consider unintended and unpredicted effects of an experimental perturbation. Our goal is a precise one, namely to silence gene expression of a measurable gene, luciferase, using RNA interference (RNAi). Each group will begin by designing a short interfering RNA (siRNA) against luciferase, but as we'll see, siRNAs can vary in efficacy and specificity. After transfecting a mammalian cell line with the siRNA you’ve designed and a reporter plasmid, we will evaluate the silencing using a luciferase assay and microarray technology. The first assay evaluates the efficacy of the siRNA in silencing. The second assay gives genome-wide expression data to reveal the specificity of your siRNA for the gene you’ve targeted. Through this combined approach, we'll assess the balance of targeted and off-target effects. | ||
[[Image:Macintosh_HD-Users-nkuldell-Desktop-sprinkler.jpg|400px|center]] | [[Image:Macintosh_HD-Users-nkuldell-Desktop-sprinkler.jpg|400px|center]] | ||
[[20.109(F07): TA's notes for module 2| TA notes, mod 2]] | [[20.109(F07): TA's notes for module 2| TA notes, mod 2]] | ||
Revision as of 09:03, 7 June 2007
Module 2
Instructors: Natalie Kuldell and Agi Stachowiak
TA:
In this experiment, we will consider unintended and unpredicted effects of an experimental perturbation. Our goal is a precise one, namely to silence gene expression of a measurable gene, luciferase, using RNA interference (RNAi). Each group will begin by designing a short interfering RNA (siRNA) against luciferase, but as we'll see, siRNAs can vary in efficacy and specificity. After transfecting a mammalian cell line with the siRNA you’ve designed and a reporter plasmid, we will evaluate the silencing using a luciferase assay and microarray technology. The first assay evaluates the efficacy of the siRNA in silencing. The second assay gives genome-wide expression data to reveal the specificity of your siRNA for the gene you’ve targeted. Through this combined approach, we'll assess the balance of targeted and off-target effects.
