20.109(F07):Module 2

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==Module 2==
==Module 2==
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'''Instructors:''' [[Natalie Kuldell]] and [[Agi Stachowiak]]
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'''Instructors:''' [[Natalie Kuldell]] and [[User:AgiStachowiak| Agi Stachowiak]]
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'''TA:'''  
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'''TA:'''[[User:AliceLo| Alice Lo]]
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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]]
[[Image:Macintosh_HD-Users-nkuldell-Desktop-sprinkler.jpg|400px|center]]
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[[20.109(F07): siRNA design | Module 2 Day 1: siRNA design and introduction to cell culture]]<br>
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[[20.109(F07): Transfection | Module 2 Day 2: Transfection]]<br>
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[[20.109(F07): Luciferase assays and RNA prep| Module 2 Day 3: Luciferase assays and RNA prep]]<br>
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[[20.109(F07): Journal article discussion| Module 2 Day 4: Journal article discussion]]<br>
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[[20.109(F07): cDNA synthesis and microarray| Module 2 Day 5: cDNA synthesis and microarray]]<br>
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[[20.109(F07): Microarray data analysis| Module 2 Day 6: Microarray data analysis]]<br>
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[[20.109(F07): Mod 2 Day 7| Module 2 Day 7: Reports due]]<br>
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[[20.109(F07): Module 2 oral presentations| Module 2 Day 8: Oral Presentations]]<br>
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direct link to [[20.109(F07): Expression engineering report| writing assignment]]<br>
[[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]]

Current revision

20.109(F07): Laboratory Fundamentals of Biological Engineering

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Module 2

Instructors: Natalie Kuldell and Agi Stachowiak

TA: Alice Lo

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.

Module 2 Day 1: siRNA design and introduction to cell culture
Module 2 Day 2: Transfection
Module 2 Day 3: Luciferase assays and RNA prep
Module 2 Day 4: Journal article discussion
Module 2 Day 5: cDNA synthesis and microarray
Module 2 Day 6: Microarray data analysis
Module 2 Day 7: Reports due
Module 2 Day 8: Oral Presentations
direct link to writing assignment

TA notes, mod 2
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