Panu Somervuo, March 19, 2007 1 cDNA microarrays.
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Transcript of Panu Somervuo, March 19, 2007 1 cDNA microarrays.
Panu Somervuo, March 19, 2007 2
cDNA microarrays• small slides with several
measurement units, spots • e.g. 2.5cm-by-7.6cm glass
slide with 30,000 spots• each spot contains specific
nucleotide sequences, probes• in hybridization process,
labeled (Cy5, Cy3) samples attach to probes
• comparative genome hybridization (CGH): DNA samples
• gene expression: RNA samples
• relative intensity of hybridization can be measured
Cy5 Cy3
Panu Somervuo, March 19, 2007 3
Data flow
• biological data, DNA/RNA extraction, fluoresence dye labeling, hybridizationarray
• scanningimage• image processing: spot segmentationdatafile
• data preprocessing and normalization:• data analysis1: statistical tests to find differentially expressed
genes gene lists
• data analysis2: biological interpretations of results
Panu Somervuo, March 19, 2007 4
Image processing• segmentation: spot signals are extracted from
background• intensity information from both spot foreground
and background• other information like spot size and shape
Panu Somervuo, March 19, 2007 7
Logarithm of ratio
• log(Cy5/Cy3) = log(Cy5) – log(Cy3)
• log2(4/1) = 2• log2(2/1) = 1• log2(1/1) = 0• log2(1/2) = -1• log2(1/4) = -2
Panu Somervuo, March 19, 2007 10
Normalization
• goal: to remove the effects of non-biological causes from data (dye-effect, hybridization, scanning, noise) and keep the biological information as well as possible
• normalization can be based on the behavior of the majority of the spots on the array, or small set of special control spots
• each normalization method is based on some assumption of the data
Panu Somervuo, March 19, 2007 11
Spot background subtraction
• how to know if spot signal is real and not just noise?• comparison against background signal• global versus local background• should background subtraction be used or not?
Panu Somervuo, March 19, 2007 12
Normalization• can be applied to both single channel and ratio data• mean
• variance
Panu Somervuo, March 19, 2007 13
Mean normalization
• global mean vs intensity dependent mean• Loess/Lowess normalization
Panu Somervuo, March 19, 2007 16
Control spots (spike-in controls)
fold change up 3log2(3)=1.58
fold change up 10log2(10)=3.32
fold change down 10log2(1/10)=-3.32
fold change down 3log2(1/3)=-1.58
Panu Somervuo, March 19, 2007 17
What is the best normalization method?
• each method is based on some assumption each method can fail
• if utilizing the behavior of majority of the spots, array should represent all genes
• if utilizing control spots, check if they are reliable
• lots of methods have been introduced, lots of methods will be introduced…
Panu Somervuo, March 19, 2007 18
Finding differentially expressed genes
• Manually set fold change cutoff• Fold change cutoff based on data• Statistical test, p-value