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The anatomy of your data files from Affymetrix array analysis .DAT= image file (107 pixels) .CEL= measured cell intensities .CDF= cell descriptions files (identify probe sets and probe set pairs) .CHP= calculated probe set data .RPT= report generated from .CHP
Chip defects
Internal controls
B. subtilis genes (added poly-A tails) Assessment of quality of sample preparation Also as hybridization controls
Hybridization controls (bioB, bioC, bioD, cre) E. coli and P1 bacteriophage biotin-labeled cRNAs Spiked into the hybridization cocktail Assess hybridization efficiency
Actin and GAPDH assess RNA sample/assay quality Compare signal values from 3 end to signal values from 5 end ratio generally should not exceed 3 Percent genes present (%P) Replicate samples - similar %P values
5. Data mining-how to interpret > 6000 measures Databases Software Techniques-clustering, pattern recognition etc. Comparing to prior studies, across platforms?
6. Validation
Experimental Design
A good microarray design has 4 elements
1. 2. A clearly defined biological question or hypothesis Treatment, perturbation and observation of biological materials should minimize systematic bias Simple and statistically sound arrangement that minimizes cost and gains maximal information Compliance with MIAME (minimal information about microarray experiment)
3.
4.
The goal of statistics is to find signals in a sea of noise The goal of exp. design is to reduce the noise so signals can be found with as small a sample size as possible
Experimental Replicates
Why?
In any exp. system there is a certain amount of noiseso even 2 identical processes yield slightly different results Sources? In order to understand how much variation there is it is necessary to repeat an exp a # of independent times Replicates allow us to use statistical tests to ascertain if the differences we see are real
As we progress from the starting material to the scanned image we are moving from a system dominated by biological effects through one dominated by chemistry and physics noise Within Affy platform the dominant variation is usually of a biological nature thus best strategy is to produce replicates as high up the experimental tree as possible
To do this, .DAT files containing array images are first processed to produce a .CEL file, which contains measured intensities for each probe on the array.
It is the .CEL files that are analyzed by the expression calling algorithm.
Every probe pair in a probe SET has a potential vote for presence call
PM and MM Probes
The purpose of each MM probe is to provide a direct measure of background and stray-signal (perhaps due to cross-hybridization) for its perfect-match partner. In most situations the signal from each probe-pair is simply the difference PM - MM. For some probe-pairs, however, the MM signal is greater than the PM value; we have an apparently impossible measure of background.
Basic analysis in MAS 5.0, but it wont handle replicates Import MAS 5.0 (.CHP) data into other software, Genesifter, GCOS, SpotFire, and many others
Signal Intensity
Following these calculations, the MAS 5.0 algorithm now has a measure of the signal for each probe in a probeset. Other algortihms, ex RMA, GCRMA, dCHIP, PLIER and others have been developed by academic teams to improve the precision and accuracy of this calculation In our Exp we will use RMA and GCRMA
List of genes that are upregulated or downregulated Determine fold up or down cutoffs
What is significant?
1.5 fold up/down? 2 fold up/down? 10 fold up/down?
Normalization is necessary to effectively make comparisons between chips-and sometimes within a single chip.
There are different methods of normalization the assumptions of where variation exist will determine the normalization techniques used.
Caveat
There is NO standard way to analyze microarray data Still figuring out how to get the best answers from microarray experiments Best to combine knowledge of biology, statistics, and computers to get answers
Venn Diagrams
MAS 5.0 GCRMA
RMA
GCRMA
Now what????
What is the first set of genes on our chips that will be filtered out?
Back to Biology
Do the changes you see in gene expression make sense BIOLOGICALLY? If they dont make sense, can you hypothesize as to why those genes might be changing? Leads to many, many more experiments
and Humans
and anything else!
GO develops a common language applicable to any organism GO terms can be used to annotate gene products from any species, allowing comparison of information across species
The ontology. Dividing human knowledge into a clean set of categories is a lot like trying to figure out where to find that suspenseful black comedy at your corner video store. Questions inevitably come up, like are Movies part of Art or Entertainment? (Yahoo! lists them under the latter.) -Wired Magazine, May 1996
Biological Examples
Biological Process Molecular Function Cellular Component
Validation
Not enough to just do microarrays Usually validate microarray results via some other technique
rt-PCR TaqMan Northern analysis Protein level analysis
No technique is perfect
First published sequence claimed 6274 genes a # that has been revised many times, why?
The Affy detection oligonucleotide sequences are frozen at the time of synthesis, how does this impact downstream data analysis?
definition: MAPKKK cascade involved in transduction of mating pheromone signal, as described in Saccharomyces definition_reference: PMID:9561267
SGD
Homework
1. Go to http://www.yeastgenome.org/ and find 3 candidate genes of known f(x) and one of undefined f(x) that you might predict to be altered by DMSO treatment What GO biological processes and molecular mechanisms are associated with your candidate genes? Where, subcellularly does the protein reside in the cell? What other proteins are known or inferred to interact with yours? How was this interaction determined? Is this a genetic or physical interaction? Find the expression of at least one of your known genes in another public ally deposited microarray data set?
1. 2. Name of data set and how you found it? What is the largest Fold change observed for this gene in the public study?
2. 3. 4.
5.
6.
Now that you are microarray technology experts can you give me 3 reasons why the observed transcript level difference may not be confirmed through a second technology like RTQPCR?
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