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.

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

TA notes, mod 2