Difference between revisions of "20.109(S09):Module 2"
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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. | 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:20.109_Control-uArray.jpg|thumb|left|400px|'''Control Microarray''' Sample expression microarray spanning entire human genome, dual colour using identical RNA. Equal red and green fluorescence intensities combine to produce yellow spots.]] | ||
| + | [[Image:20.109_Control-uArray-Zoom.jpg|thumb|center|175px|'''Control Microarray, Zoomed In''']] | ||
| + | <br style="clear:both;"/> | ||
Atissa presentation on Day 2, EHS presentation likely on Module 1 Day 8 | Atissa presentation on Day 2, EHS presentation likely on Module 1 Day 8 | ||
Revision as of 20:22, 18 December 2008
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Module 2
Instructors: Leona Samson, Agi Stachowiak
TA: Steve Goldfless
Original module developed by Natalie Kuldell and Leona Samson.
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.
Atissa presentation on Day 2, EHS presentation likely on Module 1 Day 8
Module 2 Day 1: siRNA design and introduction to cell culture
Module 2 Day 2: Journal article discussion
Module 2 Day 3: Journal Club
Module 2 Day 4: Journal Club
Note: week off between day 4 and 5 of lab.
Module 2 Day 5: Transfection
Module 2 Day 6: Luciferase assays and RNA prep
Module 2 Day 7: cDNA synthesis and microarray
Module 2 Day 8: Microarray data analysis
