Lecture 23 – Functional Genomics I Based on chapter 8 Functional and Comparative Genomics Copyright © 2010 Pearson Education Inc.

The Transcriptome – RNA Expression Analysis 1.Single Gene Approaches  Northern blotting  RT-PCR  Real-time PCR 2.Genomewide RNA expression analysis  Microarrays  SAGE  RNA Seq

Slide 3 - Southern Blot Analysis of Sequences in the Genome

2 - Northern Blot Analysis

4 - RT-PCR and mRNA Quantification 1. Isolate mRNA 2. Reverse transcribe mRNA (make a DNA copy of each mRNA) 3. PCR amplify the first strand reverse transcribed mRNAs 4. Agarose Electrophoresis of samples

5 - Real-time PCR - Review  Real-time PCR is a form of reverse transcription RCR where the method of analysis involves continuous monitoring of the PCR product formed.

The Transcriptome – RNA Expression Analysis 1.Single Gene Approaches  Northern blotting  RT-PCR  Real-time PCR 2.Genomewide RNA expression analysis  Microarrays  SAGE  RNA Seq

10 - Northern Blots vs. Microarrays 1.Global expression analysis: microarrays a.RNA levels of every gene in the genome analyzed in parallel 2.Northern blot a.Only 1 gene at a time target – loading – control

11 - Basics of Microarrays 1.DNA probe attached to solid support a.Glass, plastic, or nylon 2.RNA or cDNA is labeled a.Usually indirectly 3.Bound DNA is the equivalent of the “probe” a.Labeled RNA (cDNA) is the “target” 4.Each “probe” is specific for a different gene.

12 - Microarray Hybridization 1.Usually comparative a.Ratio between two samples 2.Examples a.Tumor vs. normal tissue b.Drug treatment vs. no treatment c.Embryo vs. adult mRNA cDNA DNA microarray samples

14 - DNA spotting I 1.DNA spotting usually uses multiple pins 2.DNA in microtiter plate 3.DNA usually PCR amplified 4.Oligonucleotides can also be spotted

17 - How Microarrays are Made: Affymetrix GeneChips 1.Oligonucleotides synthesized on silicon chip a.One base at a time 2.Uses process of photolithography a.Developed for printing computer circuits

18 - Affymetrix GeneChips 1.Oligonucleotides a.Usually 20–25 bases in length b.10–20 different oligonucleotides for each gene 2.Oligonucleotides for each gene selected by computer program to be the following: a.Unique in genome b.Nonoverlapping 3.Composition based on design rules a.Empirically derived

19 - Photolithography 1.Light-activated chemical reaction a.For addition of bases to growing oligonucleotide 2.Custom masks a.Prevent light from reaching spots where bases not wanted lampmaskchip

20 - Example: Building Oligonucleotides by Photolithography 1.Want to add nucleotide G 2.Mask all other spots on chip 3.Light shines only where addition of G is desired 4.G added and reacts 5.Now G is on subset of oligonucleotides light

21 - Example: Adding a Second Base 1.Want to add T 2.New mask covers spots where T not wanted 3.Light shines on mask 4. T added 5.Continue for all four bases 6.Need 80 masks for total 7.20-mer oligonucleotide light

23 - Target labeling: Fluorescent cDNA 1.cDNA made using reverse transcriptase 2.Fluorescently labeled nucleotides added 3.Labeled nucleotides incorporated into cDNA

25 - Labels 1.Cy3 and Cy5 a.Fluoresce at different wavelengths b.Used for competitive hybridization 2.Biotin a.Binds to fluorescently labeled avidin b.Used with Affymetrix GeneChips

Scanning of Microarrays 1.Confocal laser scanning microscopy 2.Laser beam excites each spot of DNA 3.Amount of fluorescence detected 4.Different lasers used for different wavelengths a.Cy3 b.Cy5 laser detection

SAGE 1.Seqeunce tags are PCR copied from cDNA libraries. 2.Tags are ligated together and sequenced. 3.The frequency of each TAG determines gene expression level.

14 - MPSS I 1.Massively parallel signature sequencing 2.Means of determining abundance of RNA species 3.Unique tags added to cDNAs 4.Tags hybridized to oligonucleotides on microbeads

Slide 15 – MPSS I  Sequencing performed in glass chamber  Initiated by restriction enzyme revealing four- base overhang  Hybridization of four- base adapters used to read sequence  Number of times a particular sequence is found is measure of RNA abundance