20.109(F07):Module 2

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(Module 2)
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In this experiment, we will consider  
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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.
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[[Image:Macintosh_HD-Users-nkuldell-Desktop-sprinkler.jpg|400px|center]]
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[[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 12:03, 7 June 2007

20.109(F07): Laboratory Fundamentals of Biological Engineering

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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.


TA notes, mod 2
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