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

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.

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.

Control Microarray, Zoomed In

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