Difference between revisions of "20.109(S09):Module 2"

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==Module 2==
 
==Module 2==
 
   
 
   
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'''Instructors:''' [http://web.mit.edu/be/people/samson.htm Leona Samson], [[User:AgiStachowiak| Agi Stachowiak]]
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'''Instructors:''' [http://web.mit.edu/be/people/samson.htm Leona Samson], [http://openwetware.org/wiki/User:AgiStachowiak | Agi Stachowiak]
  
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'''TA:''' [[User:Stephen_J._Goldfless| Steve Goldfless]]
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'''TA:''' [http://openwetware.org/wiki/User:Stephen_J._Goldfless | Steve Goldfless]
  
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Original module developed by [[Natalie Kuldell]] and Leona Samson.
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'''Additional development and instruction''': Peter Svensson (Samson lab, mouse gene siRNAs) and Stuart Levine (BioMicro Center, microarray analysis). Original module developed by [http://openwetware.org/wiki/Natalie_Kuldell ] and Leona Samson.
  
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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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In this experiment, we will consider direct and indirect effects of an experimental perturbation, specifically the use of RNA interference (RNAi). Each group will test two different short interfering RNAs (siRNAs) in mouse cells. We will first compare the efficacies of six different siRNAs targeting luciferase. Knockdown of this gene is readily measured using a luminescence assay and reporter plasmid. Next we will test four different commercially available siRNAs targeting mouse genes of interest. Each siRNA should knock down the gene of interest (direct effect), but is also likely to affect other genes in pathways involving the target gene (indirect effect), and may even affect expression of genes that are unrelated to the target due to some non-specific effects of the siRNA or unintentional targeting of similar sequences (off-target effect). You will perform a genome-wide expression microarray experiment and try to assess how the changes in gene expression that you see fall into these three categories. Along the way, you will learn such valuable skills as how to culture cells, do basic statistics, and analyze large data sets.
  
 
[[Image:20.109_Control-uArray.jpg|thumb|left|400px|'''Control Microarray''' Sample expression microarray spanning entire human genome, using identical RNA labeled with two different colours. The resulting equal red and green fluorescence intensities combine to produce yellow spots.]]  
 
[[Image:20.109_Control-uArray.jpg|thumb|left|400px|'''Control Microarray''' Sample expression microarray spanning entire human genome, using identical RNA labeled with two different colours. The resulting equal red and green fluorescence intensities combine to produce yellow spots.]]  
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[[20.109(S09): Microarray data analysis (Day8)| Module 2 Day 8: Microarray data analysis]]<br>
 
[[20.109(S09): Microarray data analysis (Day8)| Module 2 Day 8: Microarray data analysis]]<br>
  
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[[20.109(S09): Expression engineering report| Expression Engineering Report]]<br>
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[http://openwetware.org/wiki/20.109(S09):_Expression_engineering_report | Expression Engineering Report]<br>
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[[20.109(S09): Journal club letter| Journal Club Letter]]<br>
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[http://openwetware.org/wiki/20.109(S09):_Journal_club_letter | Journal Club Letter]<br>
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[[20.109(S09): TA notes for module 2| TA notes, mod 2]]
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[http://openwetware.org/wiki/20.109(S09):_TA_notes_for_module_2 | TA notes, mod 2]

Latest revision as of 19:33, 28 July 2015


20.109(S09): Laboratory Fundamentals of Biological Engineering

20.109(S09) frontpg.JPG

Home        People        Schedule Spring 2009        | Assignments        Lab Basics        | OWW Basics       
Protein Engineering        Expression Engineering        Cell-Biomaterial Engineering              

Module 2

Instructors: Leona Samson, | Agi Stachowiak

TA: | Steve Goldfless

Additional development and instruction: Peter Svensson (Samson lab, mouse gene siRNAs) and Stuart Levine (BioMicro Center, microarray analysis). Original module developed by [1] and Leona Samson.

In this experiment, we will consider direct and indirect effects of an experimental perturbation, specifically the use of RNA interference (RNAi). Each group will test two different short interfering RNAs (siRNAs) in mouse cells. We will first compare the efficacies of six different siRNAs targeting luciferase. Knockdown of this gene is readily measured using a luminescence assay and reporter plasmid. Next we will test four different commercially available siRNAs targeting mouse genes of interest. Each siRNA should knock down the gene of interest (direct effect), but is also likely to affect other genes in pathways involving the target gene (indirect effect), and may even affect expression of genes that are unrelated to the target due to some non-specific effects of the siRNA or unintentional targeting of similar sequences (off-target effect). You will perform a genome-wide expression microarray experiment and try to assess how the changes in gene expression that you see fall into these three categories. Along the way, you will learn such valuable skills as how to culture cells, do basic statistics, and analyze large data sets.

Control Microarray Sample expression microarray spanning entire human genome, using identical RNA labeled with two different colours. The resulting equal red and green fluorescence intensities combine to produce yellow spots.
Control Microarray, Zoomed In Both high intensity and low intensity spots are primarily yellow in colour.


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

| Expression Engineering Report
| Journal Club Letter

| TA notes, mod 2
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