Lecture 23 –Functional Genomics IBased 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

DNAmicroarray

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. Oligonucleotidesa. Usually 20–25 bases in lengthb. 10–20 different oligonucleotides for each gene

2. Oligonucleotides for each gene selected by computer program to be the following:

a. Unique in genomeb. Nonoverlapping

3. Composition based on design rulesa. 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

lamp mask chip

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 mask4. T added 5. Continue for all four

bases6. Need 80 masks for

total7. 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 Cy5a. Fluoresce at different wavelengths

b. Used for competitive hybridization

2. Biotina. 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 wavelengthsa. Cy3b. Cy5

laserdetection

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