Download presentation
Presentation is loading. Please wait.
Published byJune Banks Modified over 9 years ago
1
Idea: measure the amount of mRNA to see which genes are being expressed in (used by) the cell. Measuring protein might be more direct, but is currently harder. Measuring Gene Expression
2
Central Assumption of Gene Expression Microarrays The level of a given mRNA is positively correlated with the expression of the associated protein. –Higher mRNA levels mean higher protein expression, lower mRNA means lower protein expression Other factors: –Protein degradation, mRNA degradation, polyadenylation, codon preference, translation rates, alternative splicing, translation lag…
3
Principal Uses of Microarrays Genome-scale gene expression analysis –Differential gene expression between two (or more) sample types –Responses to environmental factors –Disease processes (e.g. cancer) –Effects of drugs –Identification of genes associated with clinical outcomes (e.g. survival)
4
Biological question Differentially expressed genes Sample class prediction etc. Testing Biological verification and interpretation Microarray experiment Estimation Experimental design Image analysis Normalization Clustering Discrimination
5
Microarray example: Biomarker identification - lung cancer Samples Genes Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.
6
60 Cum. Survival Time (months) 0.2.4.6.8 1 01020304050 Cum. Survival (Group 3) Cum. Survival (Group 2) Cum. Survival (Group 1) p = 0.002 for Gr. 1 vs. Gr. 3 Data partitioning clinically important: Patient survival for lung cancer subgroups Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.
7
Technology basics Microarrays are composed of short, specific DNA sequences attached to a glass or silicon slide at high density A microarray works by exploiting the ability of an mRNA molecule to bind specifically to, or hybridize, the DNA template from which it originated RNA or DNA from the sample of interest is fluorescently-labeled so that relative or absolute abundances can be quantitatively measured
8
Two color vs single color Bakel and Holstege. 2007. http://www.cell-press.com/misc/page?page=ETBR
9
Other applications of microarray technology (besides measuring gene expression) DNA copy number analysis SNP analysis chIP-chip (interaction data) Competitive growth assays …
10
Major technologies cDNA probes (> 200 nt), usually produced by PCR, attached to either nylon or glass supports Oligonucleotides (25-80 nt) attached to glass support Oligonucleotides (25-30 nt) synthesized in situ on silica wafers (Affymetrix) Probes attached to tagged beads
12
cDNA Microarray Design Probe selection –Non-redundant set of probes –Includes genes of interest to project –Corresponds to physically available clones Chip layout –Grouping of probes by function –Correspondence between wells in microtiter plates and spots on the chip
13
Building the chip Ngai Lab arrayer, UC BerkeleyPrint-tip head
14
http://transcriptome.ens.fr/sgdb/presentation/principle.php
15
Example dual channel cDNA array results
17
Affymetrix GeneChips Probes are oligos synthesized in situ using a photolithographic approach Typically there are multiple oligos per cDNA, plus an equal number of negative controls The apparatus requires a fluidics station for hybridization and a special scanner Only a single fluorochrome is used per hybridization
18
There may be 5,000-100,000 probe sets per chip A probe set = 11-20 PM, MM pairs Affy
19
http://www.weizmann.ac.il/home/ligivol/pictures/system.jpg
20
Interpreting Affymetrix Output Perfect Match/Mismatch Strategy Each probe designed to be perfectly complementary to a target sequence, a partner probe is generated that is identical except for a single base mismatch in its center. These probe pairs, called the Perfect Match probe (PM) and the Mismatch probe (MM), allow the quantitation and subtraction of signals caused by non- specific cross-hybridization. The difference in hybridization signals between the partners serve as indicators of specific target abundance
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.